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2.3: Stem Cells - Biology

2.3: Stem Cells - Biology


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Stem cells are cells that divide by mitosis to form either two stem cells, thus increasing the size of the stem cell "pool", or one daughter that goes on to differentiate, and one daughter that retains its stem-cell properties. How the choice is made is still unknown. However, several genes have been found whose activity prevents a daughter cell from differentiating.

Types of Stem Cells

Several adjectives are used to describe the developmental potential of stem cells; that is, the number of different kinds of differentiated cell that they can become.

  1. Totipotent cells. In mammals, totipotent cells have the potential to become any type in the adult body and any cell of the extraembryonic membranes (e.g., placenta). The only totipotent cells are the fertilized egg and the first 4 or so cells produced by its cleavage (as shown by the ability of mammals to produce identical twins, triplets, etc.). In mammals, the expression totipotent stem cells is a misnomer — totipotent cells cannot make more of themselves.
  2. Pluripotent stem cells. These are true stem cells, with the potential to make any differentiated cell in the body (but probably not those of the placenta which is derived from the trophoblast).

Figure (PageIndex{1}): Human blastocyst showing inner cell mass (top right) and trophoblast. Image used with permission (J. Conaghan).

Three types of pluripotent stem cells occur naturally:

  • Embryonic Stem (ES) Cells. These can be isolated from the inner cell mass (ICM) of the blastocyst — the stage of embryonic development when implantation occurs. For humans, excess embryos produced during in vitro fertilization (IVF) procedures are used. Harvesting ES cells from human blastocysts is controversial because it destroys the embryo, which could have been implanted to produce another baby (but often was simply going to be discarded).
  • Embryonic Germ (EG) Cells. These can be isolated from the precursor to the gonads in aborted fetuses.
  • Embryonic Carcinoma (EC) Cells. These can be isolated from teratocarcinomas, a tumor that occasionally occurs in a gonad of a fetus. Unlike the other two, they are usually aneuploid.

All three of these types of pluripotent stem cells can only be isolated from embryonic or fetal tissue. They can be grown in culture, but only with special methods to prevent them from differentiating.

In mice and rats, embryonic stem cells can also:

  • contribute to the formation of a healthy chimeric adult when injected into a blastocyst which is then implanted in a surrogate mother;
  • enter the germline of these animals; that is, contribute to their pool of gametes;
  • develop into teratomas when injected into immunodeficient (SCID) mice. These tumors produce a wide variety of cell types representing all three germ layers (ectoderm, mesoderm, and endoderm).

Using genetic manipulation in the laboratory, pluripotent stem cells can now be generated from differentiated cells. These induced pluripotent stem cells (iPSCs) are described below.

  1. Multipotent stem cells. These are true stem cells but can only differentiate into a limited number of types. For example, the bone marrow contains multipotent stem cells that give rise to all the cells of the blood but not to other types of cells. Multipotent stem cells are found in adult animals; perhaps most organs in the body (e.g., brain, liver, lungs) contain them where they can replace dead or damaged cells. These adult stem cells may also be the cells that — when one accumulates sufficient mutations — produce a clone of cancer cells.

Stem Cells for Human Therapy

The Dream

Many medical problems arise from damage to differentiated cells. Examples:

  • Type 1 diabetes mellitus where the beta cells of the pancreas have been destroyed by an autoimmune attack
  • Parkinson's disease; where dopamine-secreting cells of the brain have been destroyed
  • Spinal cord injuries leading to paralysis of the skeletal muscles
  • Ischemic stroke where a blood clot in the brain has caused neurons to die from oxygen starvation
  • Multiple sclerosis with its loss of myelin sheaths around axons
  • Blindness caused by damage to the cornea

The great developmental potential of stem cells has created intense research into enlisting them to aid in replacing the lost cells of such disorders. While progress has been slow, some procedures already show promise. Using multipotent "adult" stem cells.

  • culturing human epithelial stem cells and using their differentiated progeny to replace a damaged cornea. This works best when the stem cells are from the patient (e.g. from the other eye). Corneal cells from another person (an allograft) are always at risk of rejection by the recipient's immune system.
  • the successful repair of a damaged left bronchus using a section of a donated trachea that was first cleansed of all donor cells and then seeded with the recipient's epithelial cells and cartilage-forming cells grown from stem cells in her bone marrow. So far the patient is doing well and needs no drugs to suppress her immune system.

Using differentiated cells derived from embryonic stem (ES) cells. Phase I clinical trials are underway to assess the safety of

  • injecting retinal cells derived from ES cells
    • into the eyes of young people with an inherited form of juvenile blindness;
    • into the eyes of adults with age-related macular degeneration.
  • injecting glial cells derived from ES cells into patients paralyzed by spinal cord injuries.

The Immunological Problems

One major problem that must be solved before human stem cell therapy becomes a reality is the threat of rejection of the transplanted cells by the host's immune system (if the stem cells are allografts; that is, come from a genetically-different individual).

A Possible Solution

One way to avoid the problem of rejection is to use stem cells that are genetically identical to the host. This is already possible in the rare situations when the patient has healthy stem cells in an undamaged part of the body (like the stem cells being used to replace damaged corneas). But even where no "autologous" stems cells are available, there may be a solution: using somatic-cell nuclear transfer .

In this technique,

  1. An egg has its own nucleus removed and replaced by
  2. a nucleus taken from a somatic (e.g., skin) cell of the donor.
  3. The now-diploid egg is allowed to develop in culture to the blastocyst stage when
  4. embryonic stem cells can be harvested and grown up in culture.
  5. When they have acquired the desired properties, they can be implanted in the donor with no fear of rejection.

Using this procedure it possible to not only grow blastocysts but even have these go on to develop into adult animals — cloning — with a nuclear genome identical to that of the donor of the nucleus. The first successful cloning by SCNT was with amphibians. Later, mammals such as sheep (Dolly), cows, mice and others were successfully cloned. And in the 11 November 2007 issue of Science, researchers in Oregon reported success with steps 1–4 in rhesus monkeys (primates like us).

Figure (PageIndex{2}): Somatic cell transfer in rhesus monkeys

Their procedure:

  • Remove the spindle and thus all nuclear material from secondary oocytes at metaphase of meiosis II.
  • Fuse each enucleated egg with a skin cell taken from a male monkey.
  • Culture until the blastocyst stage is reached.
  • Extract embryonic stem cells from the inner cell mass.
  • Establish that they have the nuclear genome of the male (but mostly the mitochondrial genome of the female).
  • Culture with factors to encourage differentiation: they grew cardiac muscle cells (which contracted), and even neuron-like cells.
  • Inject into SCID mice and examine the tumors that formed. These contained cells of all three germ layers: ectoderm, mesoderm, and endoderm.
  • However, even after more than 100 attempts, they have not been able to implant their monkey blastocysts in the uterus of a surrogate mother to produce a cloned monkey.

This should reassure people who view with alarm the report in May 2013 by the same workers that they have finally succeeded in producing embryonic stem cells (ESCs) using SCNT from differentiated human tissue. The workers assure us that they will not attempt to implant these blastocysts in a surrogate mother to produce a cloned human. And their failure with monkeys suggests that they would fail even if they did try.

While cloning humans still seems impossible, patient-specific ESCs

  • could be used in cell-replacement therapy or, failing that,
  • provide the material for laboratory study of the basis of — and perhaps treatment of — genetic diseases.

Whether they will be more efficient and more useful than induced pluripotent stem cells remains to be seen.

Questions that Remain to be Answered

  • Imprinted Genes. Sperm and eggs each contain certain genes that carry an "imprint" identifying them later in the fertilized egg as being derived from the father or mother respectively. Creating an egg with a nucleus taken from an adult cell may not allow a proper pattern of imprinting to be established. When the diploid adult nucleus is inserted into the enucleated egg (at least those of sheep and mice), the new nucleus becomes "reprogrammed". What reprogramming actually means still must be learned, but perhaps it involves the proper methylation and demethylation of imprinted genes. For example, the inactive X chromosome in adult female cells must be reactivated in the egg, and this actually seems to happen.
  • Aneuploidy. In primates (in contrast to sheep, cattle, and mice), the process of removing the resident nucleus causes molecules associated with the centrosome to be lost as well. Although injecting a donor nucleus allows mitosis to begin, spindle formation may be disrupted, and the resulting cells fail to get the correct complement of chromosomes (aneuploidy).
  • Somatic Mutations. This procedure also raises the spectre of amplifying the effect(s) of somatic mutations. In other words, mutations that might be well-tolerated in a single somatic cell of the adult (used to provide the nucleus) might well turn out to be quite harmful when they become replicated in a clone of cells injected later into the patient.
  • Political Controversy. The goal of this procedure (which is often called therapeutic cloning even though no new individual is produced) is to culture a blastocyst that can serve as a source of ES cells. But that same blastocyst could theoretically be implanted in a human uterus and develop into a baby that was genetically identical to the donor of the nucleus. In this way, a human would be cloned. And in fact, Dolly and other animals are now routinely cloned this way. The spectre of this is so abhorrent to many that they would like to see the procedure banned despite its promise for helping humans. In fact, many are so strongly opposed to using human blastocysts — even when produced by nuclear transfer — that they would like to limit stem cell research to adult stem cells (even though these are only multipotent).

Possible Solutions to the Ethical Controversy

Induced pluripotent stem cells (iPSCs)

A promising alternative to the use of embryonic stem cells in human therapy are recently-developed methods of genetically reprogramming the nuclei of differentiated adult cells so that they regain the pluripotency of embryonic stem (ES) cells.

In June 2007, three laboratories reported that introducing extra copies of only 4 genes into adult mouse skin cells (fibroblasts) enables them to regain the properties of ES cells. When these cells, named induced pluripotent stem cells (iPSCs for short), were placed in mouse blastocysts, they participated in building all the tissues of the chimeric mice that resulted. (When placed in tetraploid (4n) blastocysts — unable by themselves to develop normally — embryos were formed that thus were clones of the skin cell donor.) The four genes: c-Myc, Sox2, Oct3/4, Klf4.

By 2009, several labs had succeeded in producing fertile adult mice from iPSCs derived from mouse embryonic fibroblasts. This shows that iPSCs are just a capable of driving complete development (pluripotency) as embryonic stem cells.

Reprogramming works in humans, too! Using the same four genes, the Yamanaka lab in Japan reported on 20 November 2007, that they now had reprogrammed human skin cells to become induced pluripotent stem cells (iPSCs). And the Thomson lab in Wisconsin accomplished the same thing using SOX2, OCT4, NANOG, and LIN28.

Further evidence of the remarkable role played by these few genes is the finding that during normal embryonic development of the zebrafish, the same or similar genes (SoxB1, Oct4, Nanog) are responsible for turning on the genes of the zygote. Earlier in development of the blastula, all the genes being expressed (including these) are the mother's — mRNAs and proteins that the mother deposited in the unfertilized egg. It makes sense that the same proteins that can reprogram a differentiated cell into a pluripotent state (iPSCs) are those that produce the pluripotent cells of the early embryo.

These achievements open the possibility of

  • creating cells for laboratory study of the basis of genetic diseases.
    Examples: researchers have succeeded in deriving iPSCs from
    • patients with amyotrophic lateral sclerosis (ALS, "Lou Gehrig's disease"), and then causing them to differentiate into motor neurons (the cells affected in the disease) for study of their properties;
    • the skin cells of a patient with an inherited heart disease (long QT syndrome) and causing these to differentiate into beating heart cells for study in the laboratory.
    • The Jaenisch lab reported in the 6 March 2009 issue of Cell that they have succeeded in making iPSCs (they call them hiPSCs) from fibroblasts taken from patients with Parkinson's disease. The cells were then differentiated into dopamine-releasing cells — the cells lacking in this disease. What is particularly exciting is that they accomplished this after using the Cre-lox system to remove all the genes (e.g., SOX2, OCT4, KLF4) needed for reprogramming the fibroblasts to an embryonic-stem-cell-like condition.
    • Since that report, other laboratories — using other methods — have also created iPSCs from which all foreign DNA (vector and transgenes) has been removed. Not only should such cells be safer to use in therapy, but these results show that the stimulus to reprogram a differentiated cell into a pluripotent state need only be transitory.
  • creating patient-specific cell transplants — avoiding the threat of immunological rejection — that could be used for human therapy.

    Therapy with iPSCs has already been demonstrated in mice. Three examples:

    1. The Jaenisch lab in Cambridge, MA reported (in Science, 21 December 2007) that they had successfully treated knock-in mice that make sickle-cell hemoglobin with the human βS genes (and show many of the signs of sickle-cell disease in humans) by
    • harvesting some fully-differentiated fibroblasts from a sickle-cell mouse;
    • reprogramming these to become iPSCs by infecting them with Oct4, Sox2, Klf4, and c-Myc;
    • then removing (using the Cre-lox system) the c-Myc to avoid the danger of this oncogene later causing cancer in the recipient mice;
    • replacing the βS genes in the iPSCs with normal human βA genes;
    • coaxing, with a cocktail of cytokines, these iPSCs to differentiate in vitro into hematopoietic (blood cell) precursors;
    • injecting these into sickle-cell mice that had been irradiated to destroy their own bone marrow (as is done with human bone marrow transplants). (Although the recipient mice were different animals from the fibroblast donor, they were of the same inbred strain and thus genetically the same — like identical human twins. So the procedure fully qualifies as "patient-specific", i.e., with no danger of the injected cells being rejected by the recipient's immune system.)

    The result: all the signs of sickle-cell disease (e.g., anemia) in the treated animals showed marked improvement.

    2. In the 25 July 2013 issue of Nature, a team of Japanese scientists report that they were able to manufacture three-dimensional buds of human liver cells. Their process:
    • create human iPSCs from human fibroblasts using the techniques described above;
    • treat these with the substances needed for them to differentiate in liver cell precursors;
    • culture these with a mixture of human endothelial cells and mesenchymal stem cells (to mimic the conditions that occur in normal embryonic development of the liver);
    • implant the resulting solid masses (buds) of liver-like cells into immunodeficient mice.

    The result: the implanted buds developed a blood supply and the mice began to secrete human albumin, human alpha-1-antitrypsin, and to to detoxify injected chemicals just as human livers do.

    3. Workers in the Melton lab at Harvard University reported in the 9 October 2014 issue of Cell that they had succeeded in differentiating large numbers of human beta cells from human iPSCs (as well as from human ES cells). When transplanted into diabetic mice, these cells brought their elevated blood sugar levels back down.

Let us hope that what works in mice can someday be developed into a safe therapy that will work in humans. (In the case of Type 1 diabetes mellitus, however, even patient-derived beta cells will still be at risk of the same autoimmune rejection that caused the disease in the first place.)

Despite these successes, iPSCs may not be able to completely replace the need for embryonic stem cells and may even be dangerous to use in human therapy. Several groups have found that human iPSCs contain mutations as well as epigenetic patterns (e.g., methylation of their DNA) that are not found in embryonic stem cells. Some of the mutations are also commonly found in cancer cells.

Other approaches being explored

  • ES cells can be derived from a single cell removed from an 8-cell morula. The success of preimplantation genetic diagnosis (PGD) in humans shows that removing a single cell from the morula does not destroy it — the remaining cells can develop into a blastocyst, implant, and develop into a healthy baby. Furthermore, the single cell removed for PGD can first be allowed to divide with one daughter used for PGD and the other a potential source of an ES cell line.

Figure (PageIndex{1}): Preimplantation using ES cell

  • In altered nuclear transfer (ANT) — a modified version of SCNT (somatic-cell nuclear transfer) — a gene necessary for later implantation (Cdx2 — encoding a homeobox transcription factor) is turned off (by RNA interference) in the donor nucleus before the nucleus is inserted into the egg. The blastocyst that develops
    • has a defective trophoblast that cannot implant in a uterus
    • but the cells of the inner cell mass are still capable of developing into cultures of ES cells. (The gene encoding the interfering RNA can then be removed using the Cre/loxP technique.)
  • Jose Cibelli and his team at Advanced Cell Technology reported in the 1 February 2002 issue of Science that they had succeeded inIf this form of cloning by parthenogenesis works in humans [It does! — success with unfertilized human eggs was reported in June 2007.], it would have
    • stimulating monkey oocytes to begin dividing without completing meiosis II (therefore still 2n)
    • growing these until the blastocyst stage, from which they were able to harvest
    • ES cells.
    • the advantage that no babies could be produced if the blastocyst should be implanted (two identical genomes cannot produce a viable mammal — probably because of incorrect imprinting);
    • the disadvantage that it will only help females (because only they can provide an oocyte!) (But men may have a procedure that works for them next.)
  • On 24 March 2006, Nature published an online report that a group of German scientists had been able to derive pluripotent stem cells from the stem cells that make spermatogonia in the mouse. Both in vitro and when injected into mouse blastocysts, these cells differentiated in a variety of ways including representatives of all three germ layers. If this could work in humans, it would
    • provide a source of stem cells whose descendants would be "patient-specific"; that is, could be transplanted back into the donor (men only!) without fear of immune rejection.
    • avoid the controversy surrounding the need to destroy human blastocysts to provide embryonic stem cells.
  • The 7 January 2007 issue of Nature Biotechnology reports the successful production of amniotic fluid-derived stem cells ("AFS"). These are present in the amniotic fluid removed during amniocentesis. With the proper culture conditions, they have been shown to be able to differentiate into a variety of cell types includingSo these cells are pluripotent. Although perhaps not as versatile as embryonic stem cells, they are more versatile than adult stem cells.
    • ectoderm (neural tissue)
    • mesoderm (e.g., bone, muscle)
    • endoderm (e.g., liver)

Applied to humans, none of the above procedures would involve the destruction of a potential human life.


Exercise rejuvenates quiescent skeletal muscle stem cells in old mice through restoration of Cyclin D1

Aging impairs tissue repair. This is pronounced in skeletal muscle, whose regeneration by muscle stem cells (MuSCs) is robust in young adult animals but inefficient in older organisms. Despite this functional decline, old MuSCs are amenable to rejuvenation through strategies that improve the systemic milieu, such as heterochronic parabiosis. One such strategy, exercise, has long been appreciated for its benefits on healthspan, but its effects on aged stem cell function in the context of tissue regeneration are incompletely understood. Here we show that exercise in the form of voluntary wheel running accelerates muscle repair in old animals and improves old MuSC function. Through transcriptional profiling and genetic studies, we discovered that the restoration of old MuSC activation ability hinges on restoration of Cyclin D1, whose expression declines with age in MuSCs. Pharmacologic studies revealed that Cyclin D1 maintains MuSC activation capacity by repressing TGFβ signaling. Taken together, these studies demonstrate that voluntary exercise is a practicable intervention for old MuSC rejuvenation. Furthermore, this work highlights the distinct role of Cyclin D1 in stem cell quiescence.

Figures

Extended Data Fig. 1.. Effects of voluntary…

Extended Data Fig. 1.. Effects of voluntary wheel running on muscle.

Extended Data Fig 2.. Exercise improves multiple…

Extended Data Fig 2.. Exercise improves multiple aspects of old MuSC regenerative ability.

Extended Data Fig. 3.. The exercise-induced improvement…

Extended Data Fig. 3.. The exercise-induced improvement in old MuSC activation gradually subsides after exercise…

Extended Data Fig. 4.. The exercise-induced improvement…

Extended Data Fig. 4.. The exercise-induced improvement in old MuSC activation is transferable through serum.

Extended Data Fig. 5.. Transcriptional effects of…

Extended Data Fig. 5.. Transcriptional effects of aging and exercise in quiescent MuSCs.

Extended Data Fig. 6.. Characterization of Cyclin…

Extended Data Fig. 6.. Characterization of Cyclin D1 reduction and expression in MuSCs.

Extended Data Fig. 7.. Gene sets altered…

Extended Data Fig. 7.. Gene sets altered by Cyclin D1 reduction and by aging in…

Extended Data Fig. 8.. TGFβ-Smad3 activity is…

Extended Data Fig. 8.. TGFβ-Smad3 activity is anti-correlated with Ccnd1 in MuSCs.

Extended Data Fig. 9.. TGFβ-Smad3 activity in…

Extended Data Fig. 9.. TGFβ-Smad3 activity in MuSCs with aging, Cyclin D1 deficiency, and pharmacologic…

Fig. 1 ∣. Exercise improves old muscle…

Fig. 1 ∣. Exercise improves old muscle repair and MuSC function.

Fig. 2 ∣. Exercise partly restores the…

Fig. 2 ∣. Exercise partly restores the old MuSC transcriptome and enhances Cyclin D1 expression.

Fig. 3 ∣. Exercise improves MuSC activation…

Fig. 3 ∣. Exercise improves MuSC activation through Cyclin D1.

Fig. 4 ∣. Cyclin D1 represses TGFβ…

Fig. 4 ∣. Cyclin D1 represses TGFβ signaling activity in quiescent MuSCs.


Molecular Imaging of Human Embryonic Stem Cells Stably Expressing Human PET Reporter Genes After Zinc Finger Nuclease-Mediated Genome Editing

Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging.

Keywords: PET genome editing noninvasive imaging reporter genes stem cells.


The following terms and definitions apply to this document.

2.1 Ethical committee

A specialized organization responsible for assessing and reviewing ethical issues involved in scientific research.

2.2 Informed consent

The process by which an individual or its designated legal representative voluntarily confirms willingness to donate biological material for research-related purposes, after having been informed of all aspects of the potential research that are relevant for the decision to donate.

2.3 Stem cell

Cells with the capacity for self-renewal, and which can differentiate into one or more different types of specialized functional cells.

2.4 Totipotent stem cell

Stem cells that can differentiate to form a complete and intact new organism.

2.5 Pluripotent stem cell

Stem cells that can self-renew indefinitely in vitro and possess the potential to differentiate into cells of the three embryonic germ layers, namely the endoderm, mesoderm and ectoderm.

Note: Pluripotent stem cells include but are not limited to embryonic stem cells, somatic cell nuclear transfer (SCNT)-derived embryonic stem cells, and induced pluripotent stem cells, etc.

2.6 Embryonic stem cell

The primary undifferentiated cells derived from the inner cell mass of a blastocyst or an early-stage pre-implantation embryo that can self-renew indefinitely in vitro and possess the potential to differentiate into cells of the three embryonic germ layers.

2.7 Somatic cell nuclear transfer (SCNT)-derived embryonic stem cell

Embryonic stem cells derived from the inner cell mass of a blastocyst or an embryo produced by the in vitro transfer of a donor somatic cell nucleus into an enucleated oocyte.

2.8 Induced pluripotent stem cell

Pluripotent stem cells similar to embryonic stem cells in properties but which are derived from somatic cells through artificial reprogramming by introducing genes or proteins, or via chemical or drug treatments.

2.9 Adult stem cell

Undifferentiated stem cells located within different adult tissues.

2.10 Harvest

The process of obtaining biological samples such as tissues and/or cells from donors.

2.11 Separation

The process of obtaining target cells from biological samples.

2.12 Cryopreservation

The process by which cells are maintained in an inactive state at a low temperature (<−196°C) so they can be revived later.

2.13 Resuscitation

The process whereby cryopreserved cells return to their normal growth state.

2.14 Expansion

The process of increasing the numbers of cells upon culture.

2.15 Differentiation

The process of gradually converting stem cells into a defined cell state/fate with different morphology and functional characteristics.

2.16 Stem cell bank

A legal entity or part of a legal entity that performs biobanking of different source of stem cells and their associated information.

2.17 Cell purity

The percentage of a particular cell type with defined specific biological characteristics, such as cell surface markers, genetic polymorphisms and biological activities, within a cell population.

2.18 Cell viability

The percentage of cells that are alive and metabolically normal within a cell population.

Note: Cell viability can vary over time in culture and may be measured by metabolic activities (eg, esterase activity, Thiazole blue method based on the determination of succinic dehydrogenase [MTT]), apoptosis markers, cellular redox potential, membrane potential, proliferation rate (eg, DNA content), mitochondrial function and membrane integrity, etc.

2.19 Stem cell self-renewal

The ability of stem cells to divide symmetrically, forming two identical daughter undifferentiated stem cells or divide asymmetrically, forming one daughter cell which can proceed irreversibly to a differentiated cell lineage and ultimately lead to specialized functional differentiated cells, while the other daughter cell still retains the undifferentiated characteristics of the parental stem cell.

2.20 Stem cell differentiation potential

The ability that stem cells can produce other types of cells with stably different morphologies, structures and biological functions after cell division.


Stem Cell Research Markets & Products, 2019 & 2020-2024, Featuring Competitive Analysis for 128 Global Market Participants

The possibilities arising from stem cells have resulted in great commercial interest. The purpose of this report is to reveal market dynamics affecting basic and translational research, clinical and commercial applications, and the rising demand for stem cell research products on a global basis.

Importantly, it unveils the identities of leading market competitors and presents a market size breakdown, including detailed segmentation with 5-year projections. Use it to identify emerging market opportunities, spot new product development opportunities before your competition, and make smarter decisions, faster.

Key Report Findings

  • Stem cell research applications, including priorities by segment
  • The relative demand for stem cell products, by stem cell type
  • Analysis of stem cell manufacturing technologies, including costs, risks, and the rise of contract manufacturing organizations (CMOs)
  • Analysis of market trends, including opportunities and threats
  • Competitive analysis for 128 market participants composing the global market
  • Market size determination, including detailed segmentation and 5-year projections (2019-2024)

With market competition growing increasingly fierce, ten of the largest companies selling stem cell research products are:

  • Thermo Fisher Scientific
  • BD Biosciences, a Division of Becton Dickinson (BD)
  • Merck KGaA
  • Miltenyi Biotec
  • STEMCELL Technologies
  • Lonza Group
  • Takara Bio
  • GE Healthcare Life Sciences
  • FUJIFILM CDI
  • Sigma Aldrich

Furthermore, there are many speciality vendors of stem cell research products, such as FUJIFILM CDI, ReproCELL and Ncardia (who specialize in iPSC-related products), RoosterBio (who specializes in human MSC-specific products), and Corning (who specializes in Matrigel products to support pluripotent stem cell culture and cultureware), for example.

Market Summary

The possibilities arising from stem cell characteristics have resulted in great commercial interest, with potential applications ranging from the use of stem cells in reversal and treatment of disease, to cell therapy, tissue regeneration, bioprinting, drug discovery, toxicology testing, and more.

Of interest to clinical researchers is the potential to use stem cells within regenerative medicine, and the pharmaceutical industry is integrating stem cells to conduct pharmacological testing on cell-specific tissues. There is also demand for extracellular vesicles (EVs) derived from stem cells, because they can act as intercellular messengers and may be able to exert some types of therapeutic effects.

Traditionally, scientists have worked with both embryonic and adult stem cells as research tools. While the appeal of embryonic cells has been their ability to differentiate into any type of cell, there has been significant ethical, moral, and spiritual controversy surrounding their use for research purposes. Although some adult stem cells do have differentiation capacity, it is often limited in nature, which results in fewer options for use. Thus, when induced pluripotent stem cells (iPSCs) were discovered in 2006 by Shinya Yamanaka of Kyoto University in Japan, they represented a promising combination of adult and embryonic stem cell characteristics.

iPSCs are adult cells which are isolated and then transformed into embryonic-like stem cells through the manipulation of gene expression and other methods. The first successful generation of iPSCs was via research and experimentation using mouse cells at Kyoto University in Japan. In 2007, a series of follow-up experiments were done at Kyoto University in which human adult cells were transformed into iPSC cells. Nearly simultaneously, a research group led by James Thomson at the University of Wisconsin-Madison accomplished the same feat of deriving iPSC lines from human somatic cells.

To facilitate research resulting from interest in these far-ranging applications, a large and rapidly growing market for stem cells research products has emerged. Today, well over one-hundred companies produce products to support the activities of stem cell scientists worldwide. The tools commercialized by these companies include ones to isolate, expand, culture, differentiate, and characterize stem cells, as well as technologies to enable their production at any scale.

Broad categories of stem cell research products include, but are not limited to:

  • Stem cell lines (such as iPSCs, MSCs, HSCs, NSCs, and ESCs)
  • Stem cell culture media with supplements
  • Consumables (such as antibodies, consumables in assays, cryoprotective agents and microcarriers)
  • Instruments (such as freezers, bioreactors, cytometry devices, and assay systems)
  • Stem cell services

Specifically, the following compose the majority of stem cell product sales:

  • Primary antibodies to stem cell antigens
  • Bead-based stem cell separation systems
  • Fluorescent-based labeling and detection
  • Stem cell protein purification and analysis tools
  • Tools for DNA and RNA-based characterization of stem cells
  • Isolation/characterization services
  • Stem cell culture media and reagents
  • Stem cell-specific growth factors and cytokines
  • Tools for stem cell gene regulation
  • Mechanisms for in vivo and in vitro stem cell tracking
  • Expansion/differentiation services for stem cell media and RNAi
  • Stem cell lines
  • Done-for-you or done-with-you services

Key Topics Covered

1. REPORT OVERVIEW
1.1 Statement of the Report
1.2 Executive Summary

2. STEM CELL RESEARCH: AN INTRODUCTION
2.1 Identification of Stem Cells
2.2 Species Sources of Stem Cells for Research
2.3 Culturing Stem Cell Lines
2.4 Differentiation of Stem Cells
2.5 Benefits of Stem Cell Research
2.5.1 Basic Research
2.5.2 Stem Cells in Drug Discovery
2.5.3 Disease Modeling
2.5.4 Stem Cells in Toxicity Screening

3. STEM CELLS MARKERS
3.1 Embryonic Stem Cell (ESC) Markers
3.2 Hematopoietic Stem Cell Markers
3.3 Cancer Stem Cell Markers
3.4 Osteoprogenitor Cell Markers
3.5 Neural Stem Cell Markers
3.6 Mesenchymal Stem Cell Markers
3.7 Skin Stem Cell Markers

4. STEM CELL ISOLATION PRODUCTS
4.1 Flow Cytometry in Stem Cell Isolation
4.2 Flow Cyometry Systems

5. ANTIBODIES
5.1 Primary Antibodies
5.2 Secondary Antibodies
5.3 Recombinant Antibodies
5.4 Trial Size Antibodies
5.5 Apoptosis Antibodies
5.6 Epitope Tag and Fusion Protein Antibodies
5.7 Isotype CONTrol Antibodies
5.8 Stem Cell Marker Antibodies
5.9 Pluripotent Stem Cell Markers Antibody Products
5.10 Mesenchymal Stem Cell Markers Antibody Products
5.11 Hematopoietic Stem Cell Markers Antibody Products
5.12 Neural Stem Cell Marker Antibodies
5.13 Cancer Stem Cell Marker Antibodies
5.14 Antibody Production
5.14.1 Antibody Production and Development Services
5.14.2 Monoclonal Antibody Production Services
5.14.3 Polyclonal Antibody Production Services
5.14.4 Antibody Purification Services
5.14.5 Antibody Conjugation and Labeling Services
5.14.6 Hybridoma Services
5.14.7 Recombinant Antibody Production Services
5.15 Stem Cell Factor ELISA Kits

6. STEM CELL CULTURE MEDIA
6.1 Types of Culture Media
6.2 Choice of Cell Culture Media
6.2.1 Recommended Media for Primary Cell Culture
6.3 Stem Cell Culture Media
6.3.1 Methylcellulose Media
6.3.2 Differentiation Media
6.3.3 Expansion Media
6.4 Embryonic Stem Cell Media
6.5 Hematopoietic Stem Cell Media
6.6 Mesenchymal Stem Cell Media
6.7 Neural Stem Cell Media
6.8 Media Supplements for Stem Cell Culture
6.9 Cell Storage Media

7. BIOREACTORS
7.1 Stainless Steel Fixed Bioreactors
7.2 Single-Use Bioreactors
7.2.1 Single-Use System Types
7.2.2 Main Features of Commercial Bioreactors
7.2.3 Main Differences between Single-Use and Multi-Use Bioreactors
7.2.4 Microcarriers used in Bioreactors

8. STEM CELL LINES
8.1 Use of Stem Cell Lines
8.2 Types of Stem Cell Lines
8.2.1 Embryonic Stem Cell Lines (ESC Line)
8.2.2 Adult Stem Cell Lines
8.2.3 Induced Pluripotent Stem Cell Lines (iPSC Lines)
8.3 Commercially Available Stem Cell Lines

9. STEM CELL MANUFACTURING
9.1 Institutional Manufacturing of Stem Cells
9.2 Transition to Commercial Manufacturing of Stem Cells
9.3 CMOs Involved with Stem Cell Manufacturing
9.4 Stem Cell Manufacturing Process
9.4.1 Scale-Up Methods
9.4.2 Closed Automated Systems

10. COST OF STEM CELL MANUFACTURING
10.1 Labor Cost in Manufacturing Stem Cells
10.2 Cost of Stem Cell Manufacture in a Partially-Automated Facility
10.3 Cost of Stem Cell Manufacture in a Fully-Automatic Facility

11. STEM CELL RESEARCH PRODUCTS: MARKET ANALYSIS
11.1 Global Market for Flow Cytometry Systems used in Cell Culture Studies
11.1.1 Flow Cytometry Market Share by Company
11.2 Global Market for Stem Cell Research Antibodies
11.2.1 Stem Cell Research Antibodies Market Share by Company
11.3 Market for Stem Cell Assay Products
11.3.1 Global Market for Stem Cell Assays by Product Segments
11.4 Global Market for Stem Cell Culture Media
11.4.1 Stem Cell Culture Media Market Share by Company
11.5 Global Market for Recombinant Cell Culture Supplements
11.6 Global Market for Stem Cell Cryopreservation Products
11.6.1 Market Share of Cryopreservation Products by Type
11.7 Market for Single-Use Bioreactors in Stem Cell Culture
11.7.1 Market for Single-Use Bioreactors in Stem Cell Culture by Product Segment
11.7.2 Single-Use Bioreactor Market Share by Company
11.7.3 Massive Investments in Single-Use Bioreactors
11.8 Global Market for Microcarriers used in Cell Culture
11.9 Market for Induced Pluripotent Stem Cells
11.9.1 Toxicology Assays using iPSCs
11.10 Market for Human Mesenchymal Stem Cells (hMSCs)
11.11 Global Market for Embryonic Stem Cells
11.12 Global Market for Stem Cells
11.13 Global Market for Stem Cell Research Products
11.13.1 Leaders in Consumables and Instruments used in Stem Cell Research

12. COMPANY PROFILES
12.1 3D Biotek LLC
12.2 Abbexa Ltd.
12.3 ABclonal
12.4 Abeomics Inc.
12.5 Abnova
12.6 Absolute Antibody Ltd.
12.7 Agilent Technologies Inc.
12.8 Alomone Labs Ltd.
12.9 AmProtein - China Inc.
12.10 Antigenix America Inc.
12.11 Apceth Biopharma GmbH
12.13 Applikon Biotechnology BV
12.14 AS ONE INTERNATIONAL, INC.
12.15 Assaypro
12.16 ATCC
12.17 Athersys, Inc.
12.18 Atlas Antibodies AB
12.19 ATVIO Biotech, Ltd.
12.20 Aviva Systems Biology Corp.
12.21 Becton, Dickinson and Company
12.22 Bio Basic
12.23 BioIVT
12.24 BioLegend
12.25 BioLife Solutions Inc.
12.26 Bio-Rad Laboratories, Inc.
12.27 Biorbyt Ltd.
12.28 BioSperix Ltd.
12.29 Bioss Antibodies Inc
12.30 BioVendor - Laboratorni Medicina A.S
12.31 BioVision Inc.
12.32 Bio X Cell
12.33 Bon Opus Biosciences
12.34 BosterBio
12.35 CEDARLANE
12.36 Cell Culture Technologies LLC
12.37 Cellexus Ltd.
12.38 Cell Microsystems Inc.
12.39 Cell Signaling Technology Inc.
12.40 Celltainer Biotech
12.41 ChromoTek GmbH
12.42 Cognate BioServices Inc.
12.43 Corning Life Sciences
12.44 CUSABIO Technology LLC
12.45 Cygnus Technologies
12.46 Diagenode SA
12.47 DWK Life Sciences
12.48 EMD Millipore Corp.
12.49 enQuire BioReagents
12.50 Enzo Lifesciences Inc.
12.51 EpiGentek Group Inc.
12.52 Eppendorf AG
12.53 Eurogentec, S.A.
12.54 EXBIO Praha a.s
12.55 FabGennix International Inc.
12.56 Fitzgerald Industries International
12.57 GE Healthcare Bio-Sciences
12.58 GeneTex Inc.
12.59 GenScript
12.60 Greiner Bio-One International GmbH
12.61 HemaCare Corporation
12.62 HiMedia Laboratories
12.63 HuaBio
12.64 Hycult Biotech
12.65 HyTest Ltd.
12.66 ImmuQuest Ltd.
12.67 InSphero AG
12.68 Invent Biotechnologies Inc.
12.69 InvivoGen
12.70 Irvine Scientific, Inc. (FUJIFILM Irvine Scientific, Inc.)
12.71 iXCells Biotechnologies
12.72 Kerafast, Inc.
12.73 LifeSpan BioSciences Inc.
12.74 Lonza AG
12.75 Maine Biotechnology Services (BBI Group)
12.76 MaSTherCell S.A.
12.77 MBL International
12.78 MD Biosciences (Bioproducts Division)
12.79 Miltenyi Biotec GmbH
12.80 Minerva Biotechnologies Corporation
12.81 Monash Antibody Technologies Facility (MATF)
12.82 MP Biomedicals LLC
12.83 MyBioSource Inc.
12.84 Ncardia AG
12.85 New England Biolabs
12.86 Novasep Holding SAS
12.87 NovaTeinBio
12.88 Novus Biologicals
12.89 NSJ Bioreagents
12.90 Octane
12.91 PBS Biotech Inc.
12.92 PROGEN
12.93 PromoCell GmbH
12.94 ProSci Inc.
12.95 Proteintech Group Inc.
12.96 ProteoGenix
12.97 QED Bioscience Inc.
12.98 Ray Biotech
12.99 Rev MAb Biosciences
12.100 Reddot Biotech Inc
12.101 R & D Systems
12.102 REPROCELL Inc.
12.103 RoosterBio, Inc.
12.104 RoslinCT
12.105 SAFC Biosciences Inc.
12.106 Santa Cruz Biotechnology Inc.
12.107 Sartorius AG
12.108 ScienCell Research Laboratories
12.109 Sigma-Aldrich Corp.
12.110 Signalway Antibody LLC
12.111 Sino Biological Inc.
12.112 Shanghai Korain Biotech Co., Ltd.
12.113 SouthernBiotech
12.114 StemBioSys, Inc.
12.115 STEMCELL Technologies.
12.116 Synaptic Systems GmbH
12.117 Synthecon Inc.
12.118 Takara Bio Inc.
12.119 Terumo BCT Inc.
12.120 Thermo Fisher Scientific Inc
12.121 United States Biological
12.122 Vitrolife AB
12.123 VWR International LLC
12.124 Wuxi Donglin Sci & Tech Development Co. Ltd.
12.125 Xcellerex (GE Healthcare)
12.126 YposKesi, SAS (Genethon)
12.127 Zen-Bio, Inc.
12.128 Zellwerk GmbH

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Stem cell therapy: old challenges and new solutions

Stem cell therapy (SCT), born as therapeutic revolution to replace pharmacological treatments, remains a hope and not yet an effective solution. Accordingly, stem cells cannot be conceivable as a “canonical” drug, because of their unique biological properties. A new reorientation in this field is emerging, based on a better understanding of stem cell biology and use of cutting-edge technologies and innovative disciplines. This will permit to solve the gaps, failures, and long-term needs, such as the retention, survival and integration of stem cells, by employing pharmacology, genetic manipulation, biological or material incorporation. Consequently, the clinical applicability of SCT for chronic human diseases will be extended, as well as its effectiveness and success, leading to long-awaited medical revolution. Here, some of these aspects are summarized, reviewing and discussing recent advances in this rapidly developing research field.


Stem Cells and Regenerative Medicine: Myth or Reality of the 21th Century

Since the 1960s and the therapeutic use of hematopoietic stem cells of bone marrow origin, there has been an increasing interest in the study of undifferentiated progenitors that have the ability to proliferate and differentiate into various tissues. Stem cells (SC) with different potency can be isolated and characterised. Despite the promise of embryonic stem cells, in many cases, adult or even fetal stem cells provide a more interesting approach for clinical applications. It is undeniable that mesenchymal stem cells (MSC) from bone marrow, adipose tissue, or Wharton’s Jelly are of potential interest for clinical applications in regenerative medicine because they are easily available without ethical problems for their uses. During the last 10 years, these multipotent cells have generated considerable interest and have particularly been shown to escape to allogeneic immune response and be capable of immunomodulatory activity. These properties may be of a great interest for regenerative medicine. Different clinical applications are under study (cardiac insufficiency, atherosclerosis, stroke, bone and cartilage deterioration, diabetes, urology, liver, ophthalmology, and organ’s reconstruction). This review focuses mainly on tissue and organ regeneration using SC and in particular MSC.

1. Introduction

Most of human tissues and organs do not regenerate spontaneously, justifying why cell therapy is today a significant tissue and organ repair strategy. The concept of regenerative medicine is an emerging multidisciplinary field to revolutionize the way “to improve the health and quality of life by restoring, maintaining or enhancing tissue and functions of organs.

The history of SC began in the mid nineteenth century with the discovery that some cells could generate other cells. In the beginning of the 20th century, SC were discovered when it was found that the bone marrow contained hematopoietic SC and stromal cells [1, 2]. The first successful transplant was performed by Dr. Thomas in Cooperstown, NY, in the late 1950s. The transplant involved identical twins, one of whom had leukemia, avoiding the problems associated with nontwin transplants, such as graft-versus-host disease [3]. It was not until 1968 that the first successful nontwin (allogeneic) transplant was performed. In this case, the donor was a sibling of the patient [4]. The first successful unrelated donor transplant took place in 1973 in New York when a young boy with a genetic immunodeficiency disorder received multiple marrow transplants from a donor identified as a match through a blood bank in Denmark. The first successful unrelated donor transplant for a patient with leukemia took place in 1979 at the Hutchinson Center. Since then, bone marrow transplantation expanded rapidly during the 1990s [5].

In 1998, cells from the inner cell mass of early embryos were isolated as the first embryonic stem cell lines [6, 7]. Later, in 2006, Takahashi et al. described the IPS (induced pluripotent stem cells) [8–10]. Several categories of stem cells can be used in regenerative medicine including embryonic stem cells (ESC), fetal stem cells (FSC), and adult stem cells (ASC) [11, 12]. Not all stem cells are of equal interest in terms of ability for clinical applications and are able to evolve into different specialized cells. Fetal and adult stem cells are undifferentiated cells, which can be found within fetus or in adult tissues or organs. They are able of limited self-renewal and are multipotent, which means that they can differentiate in several types of tissue cells. Although adult stem cells cannot be expanded in culture indefinitely, the use of these cells does not present ethical problems.

Multipotent SC, self-renewing, and adherent MSC, represent a small fraction of the marrow stroma [13–21]. These nonhematopoietic stromal cells are usually harvested in vitro from bone marrow but also from other tissues of mesodermal origin: fetal or neonatal tissues (umbilical cords or placenta), adipose tissue, joint synovium, dental pulp, and so forth [22–30]. MSC are characterized by their capacity of self-renewal and differentiation in different cells types (chondrocytes, endothelial cells,…). They were initially identified as progenitors able to produce colonies of fibroblast-like cells (CFU-F for colony forming units-fibroblast), to differentiate into bone or cartilaginous tissues, and to support hematopoiesis. Indeed, MSC cultivated under adapted conditions differentiate into cells of conjunctive tissues: osteoblasts, chondrocytes, tenocytes, adipocytes, and stromal cells supporting the hematopoiesis [31]. They can also differentiate into vascular smooth muscle cells, sarcomere muscular cells (skeletal and cardiac), and endothelial cells [32–36]. Recent publications even state that they can differentiate into nonmesodermal cells such as hepatocytes, neurons, or astrocytes [37–42].

MSC do not have a defined profile of surface antigen expression but there are available markers to identify them. They are mainly characterized by the expression of different antigens, CD105, CD73, CD90, Stro-1, CD49a, CD29, and CD166. On the other hand, MSC do not express antigens CD34 and CD45 (specific of the cells of hematopoietic origin), glycophorin (specific of blood cells), antigens of differentiation of the various leucocyte populations (CD14, CD33, CD3, and CD19), and HLA-DR [43–46]. The International Society for Cellular Therapy suggested a consensual definition: cells must adhere on plastic, express CD75, CD90, and CD105 and not CD34, CD45, HLA-DR, or CD11b, CD19, and are capable of differentiation into chondrocytes, osteoblasts, and adipocytes [26, 47]. Under current conditions of in vitro culture [48], the results obtained showed that the proliferation of MSC remained within the limit of Hayflick of 40 in vitro population doublings but was affected by the age of the donors [49–54]. Recent studies show that the ability of expansion and differentiation of MSC is donor-dependent. It seems that the number of MSC and their ability of in vitro differentiation and tissue regeneration in vivo decrease with age and according to the donor pathology [55]. They generally do not circulate in the peripheral blood but are resident in mesenchymal tissues [56]. Bone marrow mesenchymal stem cells (BM-MSC) can provide a support for the growth of the hematopoietic stem cells through the secretion of cytokines and through the creation of cellular interactions either directly (adhesion molecules) or indirectly (production of the extracellular matrix components). Today, nonstandardized protocols exist for their culture, differentiation, and self-renewal ability. In addition, some MSC could be more immature, without any tissue specialization, and their existence has been suspected in human [57–59].

IPS result in the acquisition of a novel state followed by the in vitro reprogramming of an adult cell after addition of selected transcription factors. The major advance in this field was performed in 2006 with the possibility of a direct reprogramming of somatic cells into pluripotent cells starting from fibroblasts [8, 9]. Generation of IPS depends on the genes used for the induction (Oct 3-4 and Sox gene family are determinant regulators for the induction process). In the course of the reprogramming, an extinction of the characteristic genes of the fibroblast, a reexpression of embryonic genes (SSEA 1 and 4), and activation of telomerase are observed. However, the efficiency of the technique is low. It is likewise necessary to underline that the IPS are exposed to a significant risk of malignant transformation due to the presence of the oncogene c-Myc used in the reprogramming. The present interest of this type of lines and its nonembryonic origin is the possibility of establishing specific lines of deficient patients for clinical research. The IPS are thus a tool for the study of the mechanisms of cell differentiation and genetic diseases and also for pharmacological screening [60].

2. Main Clinical Applications of Stem Cells

The majority of medicine specialities and different applications can benefit in the next decade from the progress in regenerative medicine: most are at experimental stages, with the exception of bone marrow transplantation. Cell therapy covers very large potentials in many clinical fields in cancer and in regenerative medicine [61–63], and more than 3,000 trials with SC are currently in progress (https://www.clinicaltrials.gov/).

Nevertheless, before SC therapeutics can be applied in the clinic, more research is necessary to understand their behaviour upon transplantation as well as the mechanisms of their interaction with the diseased microenvironment. Many authors underlined that regenerative medicine is likely to transform in the future the way we practice medicine, using pharmacological or surgical procedures. The mechanism of action of SC is still being determined. The general consensus today suggests that the most probable mechanism may be through the release of cytokines and other growth-promoting factors.

Before clinical applications, many challenges are to be solved [64]. (i) How to differentiate SC to the desired cell phenotype and which biological and environmental parameters are important during culture for differentiation? (ii) What are the best suitable cells: which precursors or differentiated cells? (iii) What are the possible immunological barriers when allogenic cells are used? (iv) What are the best biomarkers to identify pluripotent/multipotent/precursors cells? (v) What is the role of the microenvironment (scaffolds, mechanical signals)? [65] (vi) What are the bioreactive molecules such as cytokines or growth factors that can support the formation of the desired tissue? (vii) Are there potential karyotype changes during cell culture? (viii) The translation from laboratory to clinics by using good laboratory practice (GPL) could impact on cell properties? (ix) Which are the best methods to trace cells in vivo?

It is important to note that clinical applications of biotherapies are strongly controlled in Western countries. Harvesting cells or tissues of human origin can only be performed in health centers accredited by Public Authorities (in France, different regulation laws describe the procedure of authorization related to preparation, storage, and clinical use of cells and tissues). The European Regulating Authorities are also very strict about the nature of the clinical trials and about the choice of the patients. Before grafting, different points must be precised. (i) The severity degree of the pathology has to be considered. (ii) What type of grafting is planned for the patients? (iii) The site of grafting should be defined. (iv) What is the benefit for the patient? (v) What is the clinical evaluation method to investigate the functionality of the graft? (vi) Possible side effects.

2.1. Stem Cells and Cancer

Cancer SC has been for long a concept of hematology, particularly in acute myeloid leukaemia. However, more recently, research studies have described the concept of tumor initiating cells in solid tumors [66–72]. The anticancer cell therapy includes bone marrow grafting and in particular the injection of autologous or allogenic hematopoietic stem cells (HSC) CD34+. This population (CD34+) is however heterogeneous regarding its ability to generate the various lines and is the object of many research studies [73]. The graft of HSC has gained an essential place in therapeutic oncohematology [74, 75]. By 1950s, the fundamental role of hematopoietic tissue in protection against radiations was highlighted. The first clinical trials in 1959 showed the feasibility of an engraftment of allogenic marrow [3]. In 1968, the first compatible allogenic grafts HLA were successfully carried out among patients presenting severe combined deficits [4]. Then, the first cryopreserved autografts of bone marrow were reported in lymphomas. Since then, studies were pursued to improve the clinical trials and to decrease, in autologous situations, the relapses linked to the residual disease often present in the graft. Other studies aimed to prevent, in allogenic situations, the graft versus host disease [76].

Using chemical agents or specific monoclonal antibodies, ex vivo manipulations of grafts were developed to eliminate tumoral cells or T lymphocytes. By 1984, new sources of HSC have been highlighted in the peripheral and placental blood [77, 78]. That is a major step toward the development of grafts of blood HSC. The first placental blood graft was performed by Gluckman in Paris in 1998 [79]. Since 1993, banks of cryopreserved grafts of placental origin have been developed [80–82].

The use of cytotoxic T cells or NK cells, isolated and amplified in vitro, can be proposed for anticancer applications [83–87]. The use of B cells, CD4+ T cells, regulatory T cells [88, 89], and myeloid dendritic or predendritic cells producing interferon is also possible. The injection of dendritic cells for antitumoral immunization, mainly in the residual disease, but also as adjuvant therapy, is the basis of different clinical trials. However, much remains to be understood as the cells nature, their capacity to homing to specific sites (tumor, nodes), and their capacity to stimulate the immune system.

Several clinical trials have been proposed (34 in the beginning of 2014). The main applications are MSC and graft failure, graft versus host disease, and treatment of myelodysplasia [90, 91]. This point will not be developed in this review but a lot of information can be found in the literature [92–94].

2.2. Stem Cells and Tissue Regeneration

Regenerative medicine, based on the graft of tissue native cells (i.e., myocytes, chondrocytes, etc.) or SC able to differentiate into somatic cells, holds great promise if clinical hurdles can be overcome, particularly their possible tumorigenic property. This was highlighted in a case report involving a child who received fetal neural SC as a treatment for a neurodegenerative disease, but who later unfortunately developed multifocal glioneuronal tumor from transplanted neural stem cells [95]. Many studies have been published in this area in the last 20 years [96–100].

The regeneration of damaged tissues or organs implies the existence of cells able to proliferate, differentiate, and give a functional contribution to the regenerative processes. Among the possible middle-term therapeutic applications, cardiac insufficiency, atherosclerosis, osteoarticular diseases, diabetes, and liver diseases can be considered.

In regenerative medicine, four important issues have to be taken into account: (1) the choice of the reparative cells that can form a functional tissue (2) if necessary, the choice of appropriate scaffolds for transplantation (3) the role of bioreactive molecules, such as cytokines and growth factors that support the formation of the desired tissue (4) grafting and safety studies (GMP compliance). More than 3,000 clinical trials are indexed in “https://www.clinicaltrials.gov/” (mainly in USA (25%), Europe (30%), and Asia (40%)), with most of them using MSC.

2.2.1. Heart Disease

Every year in France, 10,000 new cases of serious cardiac insufficiency are detected. Heart transplants remain the only treatment for the most advanced stages but the shortage of donors and complications of immunosuppression restrict the indications. Surgical remodeling of the left ventricle only deals with the particular anatomical forms and recent negative results have led to a review of the indications. Mechanical ventricular assistance remains a temporary solution for those waiting for a transplant. There is thus a need for new treatment solutions. Xenotransplantation is not progressing since the immunological challenges are considerable and there are major safety considerations. Gene therapy and IPS are still in their infancy [27, 101] and the complexity of the mechanisms involved in heart failure does not lend itself to this therapeutic approach. Finally, cell therapy has a place, but only in patients who retain a sufficient reserve of contractile cells. The numerous trials have not made it possible to reach a conclusion at the present time [102–109].

Today more than 40 clinical trials are listed with a majority of bone marrow, Wharton’s jelly and adipose stem cells [110–113]. Histologic observations in autopsy of samples of allogenic cardiac grafts in sex mismatch showed the formation of cardiomyocytes with the receiver genotype in the myocardial tissue coming from the donor [50]. Y genotype cardiomyocytes have been shown in the myocardium of female mice that received an intravenous injection of bone marrow coming from male mice. Isotypic studies showed the homing of progenitor stem cells from bone marrow towards the lesion sites after a coronary ligation. The molecular signals leading to tissue repair are unknown. However some cytokines released during cardiac ischemia could be involved.

The treatment of myocardial infarction (MI), however, is subject to a significant constraint: the immediate availability of cells. The intracoronary injection of stem cells prepared starting from a withdrawal of bone marrow did not lead to significant improvements (3% maximum of the ejection fraction of the left ventricle). In the same manner, the intravenous injection of MSC does not give significant results. In the case of heart failure, the cell therapy turns out to be no efficient and it seems difficult today to envisage a regenerative therapy. At the end of 2007, the US based stem cell company Osiris Therapeutics completed a human trial using allogeneic SC for the treatment for heart disease. An intravenous drip was used to deliver of the shelf MSC to patients that had recently suffered a heart attack. No deaths occurred, and the treatment is now widely thought as safe [109].

Today there is no regulatory approved cell treatment for myocardial infarction, but research and clinical studies offer the hope for successful cell therapy in the next decades.

2.2.2. Peripheral Arterial Disease

Lower limb ischemia causes a decreased blood flow in the lower leg with intense pain and swelling [114]. Recently, preliminary results of a Phase I clinical trial using adult SC treatment for severe limb ischemia was presented with endothelial progenitor cells (EPC) and MSC. The cells, obtained by bone marrow aspiration, were mixed and infused into damaged vessels. According to this study, there were no adverse effects as a result of the infusions. More importantly, their patients experienced a progressive and lasting improvement in clinical parameters including walking tests, oxygen pressure, angiography, and quality of life. The use of adult SC therapy in ischemia patients would allow the development of new mature and stable capillaries. These cells have shown the property of differentiation into endothelial or smooth muscle cells but also produce a significant amount of vascular growth factors [115–120].

Harvest Technologies Corp. (MA, USA) presented some positive results from a 30-patient clinical trial of a stem cell-based treatment of critical limb ischemia (CLI). Clinical evaluation of the patients with thromboangiitis obliterans disease conducted for 12 weeks showed that the treatment had significant clinical effect. The most important finding was that more than 85% of patients were able to save their legs. Other major endpoints also showed significant improvement including quality of life assessment and individual perception of pain there was 100% reduction in the use of pain medications. Limb perfusion, as measured by TcPO2, also showed statistically significant improvement. Thirty-three percent of the patients had serious ulcers and 90% of these showed 90% or better wound closure in 26 weeks. There were no adverse events associated with the treatment.

In 2009 Pluristem Therapeutics Inc. (Haifa, Israel) began a Phase I clinical trial with a placenta-derived SC product for the treatment of CLI (end-stage of peripheral artery disease). The different trials (12 patients) generally evaluated the safety of the product in patients with CLI.

2.2.3. Ischemia Stroke

Cerebral infarct is a process, in which brain damage increases with time. Therefore the time when treatment is started is critical. At present, the only effective treatment (tissue plasminogen activator) has to be administrated very soon after the stroke [114]. In animal models, intravenous administration of hUCB cells to rats, after induction of stroke by occlusion of the middle cerebral artery, promoted the improvement of neurological function. The cells were mainly found in the cortex and the striatum of the damaged hemisphere and outside the brain, in bone marrow and spleen, and in very small amounts in muscle, heart, lungs, and liver. These authors found that some of the injected cells showed neuronal markers (NeuN2 and MAP2), astrocytic markers (GFAP), and endothelial cell markers (FVIII) [121].

Actually, IPS cells should be ideally generated without using viral vectors and without teratoma formation for being suitable for clinical use. But today the main clinical trials in topic use autologous bone marrow MSC [122–124].

2.2.4. Nervous System and Neurodegenerative Diseases

The classical notion of a renewal of adult neuronal cells is today questioned, but the therapeutic applications still remain uncertain. The spinal cord repair is currently the purpose of a great deal of work after injury [125, 126] (10 trials) with bone marrow SC. In 2010, a first study from cells derived from embryo SC producing oligodendrocytes was carried out in a volunteer in connection with the GERON Company [127, 128]. So far, no information has been given on the result of the test and the study seems to have been stopped. In France, one group is involved in a clinical study, in cooperation with a German team, using autologous bone marrow SC. Other studies should also begin in USA, Portugal, and China.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by a progressive muscle weakness that can result in paralysis and death. The multicausality of neuron death poses a considerable problem to the development of new therapeutic strategies, including cell therapy. Numerous hypotheses have been developed about the origin of ALS, but it seems that the immune system may be involved. Cell transplantation approaches in ALS remain to generate a neuroprotective environment for degenerating motor neurons by transplantation of nonneuronal cells, rather than to replace lost motor neurons. Among the cell therapy approaches tested in motor neuron disease animal models, systemic injection of human cord blood mononuclear cells has proven to reproducibly increase the life span of SOD1G93A mice, a model of familial ALS, even if only few transplanted cells were found in the damaged areas. Bigini et al. showed that human cord blood (mononuclear cells) significantly enhanced symptoms progression and prolonged survival in SOD1G93A mice and were localized in the lateral ventricles, even 4 months after administration [129]. However, hCB-MNCs were not found in the spinal cord. These results strengthen the hypothesis that the beneficial role of transplanted cells is not due to cell replacement but is rather associated with the production and release of circulating protective factors. They observed in this study that hCB-MNCs release a series of cytokines and chemokines with anti-inflammatory properties that could be responsible of the functional improvement of mouse models of motor neuron degenerative disorders. The clinical trials (25 beginning of 2014) are mainly focused on Parkinson [130] and Alzheimer [131] diseases and amyotrophic lateral sclerosis [132] with bone marrow and umbilical cord SC.

2.2.5. Bone and Cartilage [133–135]

The prevalence of osteoarthritis and degenerative joint disease will increase in the near future, driving the market for SC therapies for joints and cartilage [136]. In fact, most of the pioneering work using stem cells for bone and tendon repair was carried out in the veterinary field, mainly injuries in racehorses. Adult MSC are able to differentiate in bone, cartilage, tendon, ligament, and muscle. Today the most studied source for bone and cartilage is the bone marrow with different scaffolds. For bone the problem is more complex, when compared to cartilage, because bone is a vascularized tissue and the formation of mineralized bone matrix is not sufficient to lead to a functional tissue [137–139].

Cartilage is a mesenchymal tissue composed of one cell-type (chondrocytes), extracellular matrix (ECM), and water. Chondrocytes represent only 1-2% of cartilage volume. The cartilage ECM is composed of collagen fibers (mainly type II collagen) supporting glycoproteins and proteoglycans which have a protein core associated with glycosaminoglycan molecules such as hyaluronic acid (HA) and chondroitin sulfate. The tissue fluid, mainly water, contributes to the particular mechanical properties of cartilage and provides nutrition and exchange with synovial fluid and with extracellular fluid or other adjacent tissues. Hyaline cartilage functions with minimal friction. It demonstrates an excellent ability to provide and adaptation to compression and distributes loads on the surface of the joint [140, 141].

Because of the limited self-healing capacity of cartilage, repair of articular defects caused by degenerative joint diseases or traumatic injuries represents an open challenge and current therapies for cartilage repair are inadequate for restoring form and function [142–144]. In vitro preparation of functionally developed biocartilage substitutes is an attractive concept for future clinical treatments of cartilage injuries and degeneration. Today a FDA approved cellular-based therapy for cartilage defects uses chondrocytes [145]. In this application, autologous cells are harvested from a biopsy and expanded ex vivo to obtain a large number of cells for transplantation. Autologous expanded chondrocytes have a low risk of immune rejection but they have a tendency to dedifferentiate (loss of phenotype) in vitro. In other words, the influence of mechanical forces on cell function in vitro has been demonstrated for engineering cartilage and bones [146]. In cartilage production, dynamic mechanical stresses on chondrocytes and MSC promote differentiation and increase matrix production [147–149].

Many clinical studies performed have demonstrated the therapeutic potentials of MSC from bone marrow or adipocytes [150–153]. Wharton’s jelly MSC offer another source that has proven a chondrogenic differentiation potential and could be used in an allogenic context [46, 154–156].

While MSC therapy is promising, the incomplete understanding of their biological characteristics and function limits today the utilization of MSC in clinical application [157]. Role of growth factors, cytokines, receptors, transmembrane signalling, and adhesive proteins in MSC interactions are still elusive. In a study of the effects of MSC in a caprine model of traumatic osteoarthritis it was showed that intra-articular delivery of autologous cells to meniscectomized joints resulted in significant meniscal and regeneration and chondroprotection. In another study, subcutaneous implantation of hydroxyapatite scaffolds loaded with allogenic MSC allows cartilage obtention. The present clinical trials for cartilage repair are mainly focused on osteoarthritis 29 trials with auto- or allo-SC from bone marrow, umbilical cord, or adipose tissues [158–160].

2.2.6. Dermatology

In mammals, cell renewal on the external surface of the skin is ensured by the keratinocytes of the basement layer which divide actively and are differentiated into cells of the stratum corneum. That activity implies the existence of SC. Unlike the SC of hair follicles confined in a niche, the SC of the epidermidis are spread along the basement membrane. The main clinical trials [10] are mainly on limb ischemia in diabetic patients [161, 162].

2.2.7. Pancreas and Diabetes [163]

The cell graft appears to be an alternative to the medical treatments for pancreas diseases. The first attempt of cell therapy by grafting of islets of Langerhans was published more than ten years ago [164–167]. Other cell sources have also been proposed for pancreas and diabetes cell therapy like adult [168, 169] or fetal [170–172] MSC, embryonic stem cells [173–175], or even IPS [176, 177] because of their differentiation potential into insulin-producing cells and their immunomodulatory properties. Two studies with different therapeutic approaches are currently investigating the influence of cord blood stem cells on improving the function of pancreatic beta cells. In the first approach, children with young-onset diabetes are infused with autologous cord blood without chemotherapy. Initial results have shown that such autologous cord blood transplantation without chemotherapy has not resulted in adverse effects but has not significantly improved the situation either. All children are still dependent on administration of insulin. In the second approach, adult patients with newly diagnosed diabetes mellitus underwent nonmyeloablative chemotherapy after receiving reinfused stem cells from autologous bone marrow. Different trials on diabetes [22] type 1 or 2 are mainly performed with autologous or allogenic bone marrow or Wharton’s jelly MSC [178–181].

2.2.8. Liver Diseases

In response to a variety of chronic injuries such as hepatitis, alcohol or drug abuse, metabolic diseases, autoimmune attack of hepatocytes or the bile duct epithelium, and congenital abnormalities, liver fibrosis occurs and finally leads to hepatic cirrhosis and liver failure. Liver transplantation is the accepted treatment option for this end-stage liver diseases and acute liver failure resulting in irreversible liver dysfunction. However, it is limited by the shortage of donor organs. Moreover, it is difficult to accept such a heavy surgical treatment for some patients because of the shortage of donor organs. In fact, correction of hepatocyte functional deficiency is the prime goal of liver transplantation. There is growing evidence in support of cell therapy. As an alternative to liver transplantation, some authors tried to use hepatocytes to treat patients with liver diseases instead of liver transplantation. However, the obstacle against their clinical applications is the requirement of large number of hepatocytes that are not available from patients themselves and as well as from other donors either. Thus, it is necessary to search for a novel source of cells. SC therapy has been accepted as one of the new approaches to recolonize liver. Several studies reported the hepatocyte differentiation potential of embryonic, fetal, or adult MSC but also IPS [182–187].

As the liver contains three different cell types, which are organized in three-dimensional structures, growth and regeneration processes are highly complex. Therefore the idea of using one-type of SC leading to these three types of cells to repair liver is acceptable. Various populations of SC are under investigation in terms of their regenerative capabilities. Recently, studies showed that extrahepatic adult MSC of different origins have demonstrated their ability to express a hepatocyte-like phenotype after being differentiated in vitro. These cells which include MSC derived from bone marrow, umbilical cord, adipose tissue, and placenta are used in 32 trials mainly for cirrhosis (after hepatitis, alcohol abuse, and liver transplantation) [188–191].

2.2.9. Urology and Erectile Dysfunction

The group of Atala in USA performed urethra transplant in young patients, prepared in vitro from bladder cells cultivated on a collagen and polyglycol acid matrix [192].

Recently, the SC therapy for erectile dysfunction has been investigated. Transplantation of SC (adipose-derived stem cells or bone marrow stem cells…) was performed by intracavernous injection [193, 194]. More recent studies used combinatory therapy by supplementing stem cells with angiogenic proteins. The different studies reported better erectile function after SC mainly by intracavernous injection [195].

The main potential applications are postprostatectomy and postradiotherapy, diabetes associated erectile dysfunction, and Peyronie’s disease [196]. Human clinical trial of erectile dysfunction with SC is not yet approved as treatment but one clinical trial in Korea was published in 2010 and two preclinical trials have been approved in USA and France.

2.2.10. Retina

The different ophthalmologic treatments with SC (mainly bone marrow) are related to retina diseases, macular degeneration, glaucoma, and hereditary dystrophy [197–199].

2.2.11. Hematology: Preparation of Red Blood Cells

In vivo production of red blood cells (RBC) can be of a great practical interest. Recently, RBC preparation has been possible in vitro based on CD34+ stem cells [200, 201]. The protocol is in three steps: (1) proliferation and induction of the erythroid differentiation, (2) culture on a model reproducing the physiological microenvironment with mesenchymal cells, and (3) culture in the presence of stromal cells alone and without any growth factor. With this protocol, the cells undergo the various phases of differentiation to red cells. The industrial development would require developing suitable bioreactors. Another solution would be to have a permanent and unlimited supply of blood products. A first option is to use embryonic SC whose differentiation gives: first CD34+ stem cells, then erythrocytes [202]. Another approach consists in using induced pluripotent SC or IPS [203]. Different lineages of IPS and/or embryonic SC are currently used experimentally beyond the difficulties in controlling complete differentiation, one major issue to be solved is that of insufficient yields [204].

2.3. Stem Cells and Whole Organ Engineering

The relevance of research into the creation of reconstructed organs is justified by the lack of organs available for transplantation and the growing needs for an ageing population. On a technical level, the development of these reconstructed organs involves two complementary stages: decellularization of the target organ with a need to maintain the structural integrity of the extracellular matrix and recellularization of the matrix with stem cells or resident cells [205, 206].

Whole organ engineering like liver, kidneys, heart, or lung is particularly difficult because of the structural complexity and heterogeneity of organ and cell types. But new ways of researches are currently focused on the matrix to support recellularization and a promising approach is the direct use of extracellular matrix of the whole organ. Thus rodent, porcine, and rhesus monkey organs have been decellularized to obtain a scaffold with preserved extracellular matrix and vascular network.

Decellularization can be achieved through an intra-arterial infusion of a solution containing the detergent Triton X-100 and ammonium hydroxide. This method causes all the cellular elements to disappear, leaving elements of the extracellular matrix and the vascular system. Other methods of decellularization have also been used, employing other chemicals, enzymes, or physical ways (ultrasounds) [207, 208].

Several types of cell can be considered for recellularization purposes: SC (embryonic, fetal, and adult SC) or the patient’s autologous cells. SC probably represent the ideal source of material due to their ability to proliferate. Their use appears to be limited, nevertheless, by their allogenic nature, which could possibly trigger an immune response and consequent rejection, in addition to the risk with ESC of the formation of teratomas in vivo. Fetal cells conserve their ability to proliferate and are easily differentiated without running the risk of induction of teratomas in vivo. These obstacles could be removed in future by using nuclear transfer techniques from the patient’s somatic cells (IPS). Finally, the stem or progenitor cells present in most organs are another source of cells that could be used for in vitro organogenesis. But, they often remain difficult to define, isolate, and grow in culture.

Furthermore, the type and number of cells to be used for recellularization vary depending on the organ to be reconstructed. Apparently, specific cells of the organ to be reconstructed are indispensable. Other types of cell, such as endothelial cells and fibroblasts, are also needed, since they promote the functional cell phenotype and contribute to the structural organization of tissue. The matrix of the vascular system of the organ to be reconstructed needs to be reendothelialized so as to orientate the blood flow and prevent thrombosis.

Currently, growing organs in vitro and ex vivo can take several weeks until they have completely developed in the matrix. For seeding the use of an extracorporeal pulsating or continuous infusion system (bioreactor) is indispensable for providing the cells with an oxygen supply and keeping the infusate at a constant temperature [209, 210]. The infusion liquids are derived from the culture media used for the cells in question. They need to contain growth factors or other molecules that are more specific to each organ. Finally, there is another hypothetical possibility for recellularization, the transplanting of a decellularized organ into the recipient, in the hope that recellularization will occur directly from the recipient’s own cells.

Encouraging work has recently shown the feasibility of creating bioorgans for the reconstruction of heart, lungs, liver, and kidneys. Clinical applications still remain a distant prospect, however.

2.3.1. Heart

Heart construction could be an alternate option for the treatment of cardiac insufficiency in the future. It is based on the use of an extra-cellular matrix coming from an animal’s heart and seeded with cells likely to reconstruct a normal cardiac function. Though the decellularization techniques now seem to be under control, the issues posed by the selection of the cells capable of generating the various components of cardiac tissue are not settled yet. In addition, the recolonization of the matrix does not only depend on the phenotype of cells that are used but also impacted by the nature of biochemical signals emitted. The complexity of those problems results in the full replacement of the heart with a biomaterial substitutes to standard transplanting is one prospect [211]. However, it is more realistic to hope, in the medium run, partial replacements of the heart with recellularized matrices reinforcing portions of the failing myocardium or with direct cellular therapy with SC.

The decellularization of animal hearts (rats and, more recently, hearts of large mammals) has been performed by D. Taylor through the infusion of chemical detergents. This study shows that the integrity of the matrix (collagen, fibronectin, laminin, fiber orientation, etc.) can be maintained as well as the permeability of the vascular tree and the competence of the heart valves [212].

Recellularization is more difficult due to the diversity of the cell populations that need to be reconstituted. Three ways to achieve this goal can be considered. (i) Use of a single population of pluripotent cells is capable of giving rise to all types of heart cells through the effect of environmental signals (an approach that appears currently to be rather unrealistic). (ii) Use of adult cells already differentiated for target lineages. The obtaining of fibroblasts and vascular cells can be achieved, especially as they can be taken from a future “recipient” of the reconstituted organ, as has been successfully demonstrated in the creation of implantable blood vessels. (iii) The third intermediate strategy consists of using a single population of progenitor cells at the mesodermic stage that would be liable, depending on the signals produced by the host tissue, to achieve differentiation in situ in the three main cell types (cardiomyocytes, endothelial cells, and smooth muscle cells). The problem of obtaining cardiogenic cells is also more complex since they not only need intrinsically contractile properties, but they must also be capable of coupling and modulating their frequency in response to neurohumoral or pharmacological stimuli. The plasticity of adult somatic cells is limited however, it does not allow them to differentiate into cardiomyocytes. This property is only possessed by pluripotent cells, capable of acquiring a cardiac phenotype under the influence of the appropriate signal inducers. Such pluripotent cells could be human embryonic stem cells (hESC) whose allogenic character poses the problem of rejection (to say nothing of the ethics debate) or autologous, adult somatic SC rendered pluripotent through reprogramming (IPS). Regardless of the origin of such pluripotent cells, however, their clinical use implies an in vitro differentiation stage and then a selection process so that only the cardiogenic progenitors would be used. More recently, a direct conversion of the adult cells (fibroblasts) into cardiomyocytes has been proposed, again passing through the pluripotent cell stage. This approach still seems to be remote for clinical applications [213].

An important challenge is the transfer of cells into the matrix to recolonized [214]. While cell infusion destined for the vascular system appears to be logical for the endothelium, intramural injection of cells for cardiogenic purposes is less obvious.

In summary, by the complete replacement of a human heart by another heart constituted from a matrix of animal origin and seeded by cells capable of providing the organ with effective, mechanical activity remains a remote prospect and is unlikely to become a reality within the next 10 to 20 years.

Another strategy for cardiac repair is the preparation of cardiac patch [215, 216]. The construction of the high biocompatible biomaterials pretreated with SC will offer a promising method to improve the effects of SC therapy for myocardial infarction. Thus the development of this cardiac SC patch has high therapeutic perspectives for the treatment of the disease and prevention of the chronic heart failure. However the materials suitable for the treatment of MI need to have specific quality: biocompatibility, resistant to the mechanical force in situ, suitable for the cell amplification, and being with suitable size of pores for the cell communication which is necessary for the formation of the functional tissue. Under microscope, the pore size needs to be at least 50 μm which is necessary for the vascularization of the patch and assure the MSC metabolism. The biological materials have more advantages than artificial materials because the integration of the cells is optimal for the construction of the cardiac SC patch. As the MSC derived from Wharton’s jelly are easy to collect, the umbilical artery can be collected at the same time. The natural matrix of the umbilical artery possesses the essential property for the construction of a biocompatible cardiac patch.

2.3.2. Lungs

About fifty million people throughout the world are living with chronic respiratory failure at a terminal stage. The only treatment for this disease that seriously reduces life expectancy is, in selected cases, lung transplantation, but the results still are poor.

A tracheobronchial graft remains a challenge [217–221]. Research has not yet found an ideal cell substitute for the airways of the lung. Failures have been observed with synthetic prostheses, bioprostheses, tracheal allografts, and autografts. In fact, not only epithelial tissue regeneration but also even cartilaginous regeneration has been observed. Research seems to indicate that this regeneration of tracheal tissue might be possible from an aortic matrix and SC taken from bone marrow [217]. Studies have been performed in humans in the context of extended cancer of the trachea and conservation surgery in cases of lung cancer. The research has also contributed to better understand tissue regeneration mechanisms [222].

Pulmonary regeneration using SC is more complex [223]. In fact, several types of local progenitor cells that contribute to cell repair have been described at different levels of the respiratory tract. Moving towards the alveolus, one finds bronchioloalveolar SC as well as epithelial cells and pneumocytes. In the category of “local SC,” cells of the subpopulation have been identified that are differentiation markers which in vitro mimic stromal mesenchymal cells. The role of these cells in tissue repair has been demonstrated in animal models. Recently it was described that resident, multipotent pulmonary SC are capable of self-renewal as well as clonogenicity. The phenotype and functional characteristics of these new cells have been specified in vitro and in vivo.

The lung also contains resident specific MSC that have been described and characterised [224–226]. These cells do not play a direct part in epithelial renewal but establish communication with the epithelium, thus ensuring their role as a local cytoprotector [227].

Finally, numerous studies performed on animals have shown a beneficial role played by exogenous MSC produced by bone marrow. The effects observed in lesional pulmonary edema, sepsis, pulmonary hypertension, and even idiopathic pulmonary fibrosis have resulted in clinical applications that are currently being assessed [228–231]. The immunomodulatory, anti-inflammatory, antiapoptotic, and angiogenic properties of MSC today place these cells at the heart of tissue repair. Contrary to past hypotheses, these cells do not seem to differentiate themselves into alveolar epithelial cells and their method of action would involve paracrine mechanisms, not all of which have as yet been explained.

With respect to the creation of a bioartificial lung,recent works on the subject have been realized with decellularized rat lung in order to obtain a supporting matrix [232]. Epithelial and endothelial cells were then injected into a pulmonary matrix followed by a five-day incubation period in a bioreactor. Morphological studies found an aspect closely resembling the animal’s own lung with respect to the alveolar cells (volume, number, and size) and in vitro physiological studies also showed that the ventilatory capacities and gas exchanges had also been maintained. An in vivo implantation of the bioartificial lung produced spontaneous ventilation for six hours. After this, pulmonary edema occurred. Several research routes, such as improvement in differentiation and maturation of the injected cells, a longer incubation period in the bioreactor and optimization of postoperative ventilation have been proposed.

Recently, others authors also decellularized a rat lung using chemical treatment, retaining only the framework matrix of the lung. This decellularized lung was then placed in a bioreactor that was used to mime the physiological conditions (negative pressure and pulsatile vascular perfusion). Epithelial cells from new-born rats were injected through the trachea and endothelial cells were injected into the vascular system. After four to eight days of incubation, this biolung was grafted on to a rat [221]. The compliance measurements were substantially different between the native lung, the decellularized lung, and the lung produced by bioengineering, with greater opening pressures reflecting a less functional surfactant in the bioengineered lung. Yet there was nothing to indicate rigidity of the matrix, thus ruling out the development of fibrosis, and gaseous exchanges were covered, attesting to the functional nature of this lung.

The decellularization of lungs was then reproduced on the lungs of pigs, nonhuman primates, and even humans [233–235]. Embryonic SC or MSC were used to cellularize decellularized lungs [235–237].

This initial research opens up a promising route for developing a functional bioartificial lung, with the prospect of application to humans within 15 to 20 years [238]. However many questions remain to be answered: is the use of a decellularized pulmonary matrix the only possible solution? Which cells should be chosen for recellularization, MSC or resident pulmonary cells? What is the optimal incubation time in a bioreactor? Would the technique be applicable to the human lung with its very extensive alveolar surface?

2.3.3. Liver

Recent researches have shown that it was possible to use decellularized liver treated by detergents as scaffold, which keeps the entire network of blood vessels and the ECM [239]. The intact blood vessel network will mimic the circulation in organ and provide appropriate oxygen and nutrient supply for bioartificial liver [240]. The ECM is composed of a complex mixture of molecules and arranged in three-dimensional spatial organization that support the cell seeding, growth, and differentiation. Decellularized liver keeps the texture of the original organ. This natural structure can provide a three-dimensional matrix in favor of cell proliferation, differentiation, and function, which promotes the emergence of the idea to use decellularized organ in bioengineering liver. The liver decellularization is carried out by perfusing detergents like Triton X-100 or sodium dodecyl sulfate, via the portal vein [241]. This method can destroy cell membrane and take off debris of cells and at the same time keeps the extracellular matric complete with blood and biliary vessels. This matrix maintains the liver-specific proteins proportions for collagens I and IV, fibronectin, and laminin. The intact vascular system is useful for recellularization.

Besides decellularized whole organ scaffold, the choice of cells selected to repopulate a decellularized liver scaffold is critical to the function of bioengineered liver. At present, potential cell sources are hepatocyte and MSC. SC, such as liver stem cells, ESC, IPSCs, and MSC, are a promising alternative for primary hepatocytes. Recent studies have shown that MSC originated from extrahepatic tissues can differentiate into endoderm cell-lines as hepatocytes. Several methods have successfully differentiated MSC into hepatocytes, such as stimulation MSC by cytokines as growth factors direct and indirect coculture of MSC with hepatocytes, or promotion of MSC differentiation in a three-dimensional matrix. In some cases, differentiation of MSC into hepatocytes can also be an alternative approach for whole organ transplantation in treatment of acute and chronic liver diseases [183]. Moreover, it has been shown that decellularized liver scaffold supports hepatic differentiation of MSC, leading to cells with morphological and functional characteristics of mature hepatocytes [242, 243].

2.3.4. Kidney [244–246]

The kidney is certainly one of the most difficult organs to reconstruct due to its tissue complexity and the heterogeneous nature of the cells from which it is constituted. There is relatively few researches on kidney autoconstruction, though experiments performed on rats, pigs, and monkeys [247]. The first demonstration of the feasibility of the technique was provided by Ross in the rat [248]. They seeded the decellularized organ with pluripotent murine embryonic stem cells antegrade through the artery or retrograde through the ureter. The cells introduced were differentiated into glomerular, tubular, and vascular structures. They nevertheless lost their embryonic phenotype as it could be seen from the appearance of immunohistochemical markers. Nakayama et al. [249] decellularized sections of the kidneys taken from macaques at various growth stages from fetus to adult, via intermediate ages, with the aim of optimizing decellularization techniques and recellularization in vitro [249, 250]. They demonstrated that the appearance of Pax-2 and vimentin markers after the cells had been implanted originated from the kidneys of the fetus.

As with other organs, the research into the construction of a kidney raises numerous questions about the preparation of a matrix and the sources of the cells used for recellularization. Biological matrices have proved their superiority over the synthetic matrices sometimes used in tissue engineering. In the case of the kidney, the most frequently employed matrices are allogenic, even though xenogeneic matrices can be considered, although they might be subject to specific immunological and regulatory issues.

In summary, a large number of questions and problems remain to be solved before a kidney can be prepared or constructed from ECM. Furthermore, none of the “self-constructed” organs in animals has proved to be capable of performing the vital function in the recipient for longer than a few hours. In the case of the kidney, no transplant has yet been reported even though it is the main challenge for research. The objective remains plausible, however, even if clinical applications appear to be very remote, certainly not before 15 to 20 years.

3. MSC Secretome for Tissue Repair: Towards a Cell-Free Therapy

Even if initially MSC were proposed for cell therapy based on their differentiation potential, the lack of correlation between functional improvement and cell engraftment or differentiation at the site of injury has led to the proposal that MSC exert their effects not through their differentiation potential but through their secreted product [251, 252]. The secretion of bioactive factors is then thought to play a predominant role in the mechanisms of action of MSC. Haynesworth et al. [253] were the first to report that MSC synthesize and secrete a broad spectrum of growth factors, chemokines, and cytokines that could exert significant effects on cells in their vicinity. Since that, many researches have been focused on the characterization of the MSC secretome, including both soluble factors and factors released in extracellular vesicles (e.g., exosomes and microvesicles) and their therapeutic potential [254–256].

The results from most investigations show that MSC-conditioned medium or its components mediate some biological functions of MSC. Several studies have reported that MSC-derived exosomes have functions similar to those of MSC, such as repairing tissue damage, suppressing inflammatory responses, modulating the immune system, or even decreasing cancer cells proliferation [257–264].

Together these studies provided pivotal support for the paracrine hypothesis such that MSC therapy is increasingly rationalized on MSC secretion rather than its differentiation potential. However, the mechanisms are still not fully understood and the results remain controversial. Compared with cells, exosomes are more stable and reservable, have no risk of aneuploidy, a lower possibility of immune rejection following in vivo allogeneic administration, and may provide an alternative therapy for various diseases.

4. Conclusions

The regeneration of tissues and organs and the use of SC for clinical uses are and will remain a challenge for the development of cell therapy and tissue engineering. Fetal and adult SC and in particular MSC provide exciting therapeutic tools of regenerative medicine. However basic research should be developed to better understand the biological process and molecular mechanism of SC differentiation, as well as the role of the mechanical signals.

Several challenges should be overcome: (i) increase of the yield of preparation of the differentiated stem cells and study of the heterogeneous character of the preparations (ii) possibility to have a standardized and reproducible product (preparation of controlled batches) (iii) technical problems regarding the definition of scaffolds, cells used, long-term stability, and culture medium. In particular, the impact of the biomaterial used remains to be defined (iv) grafting (biotissue can be introduced via direct cell implantation (cell therapy), biotissue transplantation, or gene therapy) (v) risk of teratogenic effect and of immune reaction (i.e., in the umbilical cord cells the immune risk being weaker) (vi) religious and legal issues with respect to the different country regulations.

Current knowledge allows optimism for the future but definitive answers can only be given after long-term randomized and controlled clinical trials.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this review.

Authors’ Contribution

J.-F. Stoltz and N. de Isla contributed equally.

Acknowledgments

The Région Lorraine, French embassy in Beijing and Wuhan French Consulate are acknowledged for their financial support of this paper.

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Copyright

Copyright © 2015 J.-F. Stoltz et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Sleeper cells: Newly discovered stem cell resting phase could put brain tumors to sleep

By analyzing brain stem cell data, Assistant Professor Christopher Plaisier and biomedical engineering doctoral student Samantha O'Connor saw the phases of the cell cycle mapped out in more detail than previously possible -- the G0 resting phases, including a new, separate phase they called Neural G0, in addition to growth phases G1 and G2, copying phase S and splitting phase M. Credit: Christopher Plaisier/ASU

Christopher Plaisier, an assistant professor of biomedical engineering in the Ira A. Fulton Schools of Engineering at Arizona State University, and Samantha O'Connor, a biomedical engineering doctoral student in the Plaisier Lab, are leading research into a new stage of the stem cell life cycle that could be the key to unlocking new methods of brain cancer treatment. Their work was recently published in the research journal Molecular Systems Biology.

"The cell cycle is such a well-studied thing and yet here we are looking at it again for the umpteenth time and a new phase pops out at us," Plaisier says. "Biology always has new insights to show us, you just have to look."

The spark for this discovery came through a collaboration with Patrick Paddison, an associate professor at the Fred Hutchinson Cancer Research Center in Seattle, and Dr. Anoop Patel, an assistant professor of neurological surgery at the University of Washington who is also involved in the Fred Hutchinson Cancer Research Center.

Paddison's team called upon Plaisier to help analyze their brain stem cell data characterized through a process called single-cell RNA sequencing.

"That data turned out to be pretty amazing," Plaisier says. "It mapped out into this beautiful circular pattern that we identified as all of the different phases of the cell cycle."

O'Connor developed a new cell cycle classifier tool—called ccAF, or cell cycle ASU/Fred Hutchinson to represent the collaboration between the two institutions—that takes a closer, "high-resolution" look at what's happening within the growth cycles of stem cells and identifies genes that can be used to track progress through the cell cycle.

"Our classifier gets deeper into the cell cycle because there could be pieces we're capturing that have important implications for disease," O'Connor says.

When Plaisier and O'Connor used the ccAF tool to analyze cell data for glioma tumors, they found the tumor cells were often either in the Neural G0 or G1 growth state. And as tumors become more aggressive, fewer and fewer cells remain in the resting Neural G0 state. This means more and more cells are proliferating and growing the tumor.

They correlated this data with the prognosis for patients with glioblastoma, a particularly aggressive type of brain tumor. Those with higher Neural G0 levels in tumor cells had less aggressive tumors.

They also found that the quiescent Neural G0 state is independent of a tumor's proliferation rate, or how fast its cells divide and create new cells.

"That was an interesting finding from our results, that quiescence itself could be a different biological process," Plaisier says. "It's also a potential point where we could look for new drug treatments. If we could push more cells into that quiescent state, the tumors would become less aggressive."

Current cancer drug treatments focus on killing cancer cells. However, when the cancer cells are killed, they release cell debris into the surrounding area of the tumor, which can cause the remaining cells to become more resistant to the drugs.

"So, instead of killing the cells, if we put them to sleep it could potentially be a much better situation," Plaisier says.

With their ccAF tool, they were also able to find new states at the beginning and end of the cell cycle that exist between the commonly known states. These are among the topics for their next phase of research.

"We're starting to think about ways to dig into those and learn more about the biology of the entry and exit from the cell cycle because those are potentially really important points where the cells will either go into the G1 state or G0," Plaisier says.

Figuring out what triggers a cell to enter the division cycle or remain in a G0 resting state could help understand the processes behind tumor growth.

"The primary feature of any cancer is that the cells are proliferating," Plaisier says. "If we could get in there and figure out what the mechanisms are, that might be a place to slow them down."

Plaisier and O'Connor are making the ccAF classifier tool open source and available in a variety of formats for anyone studying single-cell RNA sequencing data to ease into the process of studying cell cycles.


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Repair of the heart is an old dream of physicians caring for patients with cardiac disease. Experimental studies suggest that cardiac transfer of stem and progenitor cells can have a favorable impact on tissue perfusion and contractile performance of the injured heart. Some researchers favor stable stem cell engraftment by fusion or transdifferentiation into cardiomyocyte or vascular cell lineages as likely explanations for these beneficial effects. Others have proposed that transient cell retention may be sufficient to promote functional effects, eg, by release of paracrine mediators. Although the mechanistic underpinnings of stem cell therapy are still intensely debated, the concept of cell therapy has already been introduced into the clinical setting, where a flurry of small, mostly uncontrolled trials indicate that stem cell therapy may be feasible in patients. The overall clinical experience also suggests that stem cell therapy can be safely performed, if the right cell type is used in the right clinical setting. Preliminary efficacy data indicate that stem cells have the potential to enhance myocardial perfusion and/or contractile performance in patients with acute myocardial infarction, advanced coronary artery disease, and chronic heart failure. The field now is rapidly moving toward intermediate-size, double-blinded trials to gather more safety and efficacy data. Ultimately, large outcome trials will have to be conducted. We need to proceed cautiously with carefully designed clinical trials and keep in mind that patient safety must remain the key concern. At the same time, continued basic research to elucidate the underlying mechanism of stem cell therapy is clearly needed.

The dogma of the heart as an organ composed of terminally differentiated myocytes incapable of regeneration is being challenged. Evidence has been presented that a fraction of cardiomyocytes may be able to reenter the cell-cycle and that limited regeneration can occur through recruitment of resident and circulating stem cells. 1 Some clinicians may regard these new ideas as being mere curiosities, because of their everyday experience that endogenous repair mechanisms are overwhelmed in patients with acute myocardial infarction (AMI), advanced coronary artery disease, and chronic heart failure. However, the existence of endogenous repair mechanisms suggests that cardiac repair may be achieved therapeutically in these clinical settings. Evidence to support this hypothesis will be reviewed in this article.

Another concept that has recently generated excitement by some, but disbelief by others, is the concept of adult stem cell plasticity. 2,3 Stem cells are capable of self-renewal, transformation into dedicated progenitor cells, and differentiation into specialized progeny. Traditionally, tissue-resident adult stem cells were believed to differentiate into progeny only within tissue lineage boundaries. Plasticity implies that stem cells can transdifferentiate into mature cell types outside their original lineage in response to microenvironmental cues. For example, hematopoietic stem cells (HSCs), when transplanted into the (murine) myocardium, may transdifferentiate into cardiomyocytes and blood vessels, thereby improving heart function and survival. 4

Ironically, although cell therapy is already being introduced into the clinical setting, fusion of transplanted stem cells with resident cardiomyocytes has been offered as an alternative explanation for previous claims of transdifferentiation. 5,6 Moreover, the mechanistic underpinnings of stem cell therapy appear to be far more complex that previously anticipated. It has been proposed that stem cells release angiogenic ligands, protect cardiomyocytes from apoptotic cell death, induce proliferation of endogenous cardiomyocytes, and may recruit resident cardiac stem cells (Figure). 7–11 Regardless of the mechanisms, there appears to be general agreement that stem cell therapy has the potential to improve perfusion and contractile performance of the injured heart. 4,7–9,11,12

Working hypothesis of therapeutic stem cell transplantation for myocardial regeneration. Stem and progenitor cell transplantation can have a favorable impact on tissue perfusion and contractile performance by promoting vascularization and myocyte formation. Improved vascularization may facilitate beneficial effects in the myocyte compartment. Depending on the stem cell type and local milieu, the relative contribution of cell incorporation (transdifferentiation and/or fusion) vs paracrine effects may vary. Stem and progenitor cell numbers and functional capacity are influenced by a patient’s age, gender, cardiovascular risk factors, and underlying disease state (see text for details).

Potential Donor Cells

Conceptually, a variety of stem and progenitor cell populations could be used for cardiac repair. Each cell type has its own profile of advantages, limitations, and practicability issues in specific clinical settings. Studies comparing the regenerative capacity of distinct cell populations are scarce. Many investigators have therefore chosen a pragmatic approach by using unfractionated bone marrow cells (BMCs), 13–24 which contain different stem and progenitor cell populations, including HSCs, endothelial progenitor cells (EPCs), and mesenchymal stem cells (MSCs). Ease of harvest and lack of extensive requirement for ex vivo manipulation are additional advantages of using unselected BMCs.

Endothelial Progenitor Cells

EPCs have originally been defined by their cell surface expression of the hematopoietic marker proteins CD133 and CD34 and the endothelial marker vascular endothelial growth factor receptor-2, and their capacity to incorporate into sites of neovascularization and to differentiate into endothelial cells in situ. 25 Increasing evidence suggests that culture-expanded EPCs also contain a CD14 + /CD34 − -mononuclear cell population with “EPC capacity,” which mediates its angiogenic effects by releasing paracrine factors. 26,27 Notably, EPC numbers and their angiogenic capacity are impaired in patients with coronary artery disease, which may limit their therapeutic usefulness. 28,29

CD133 + Cells

The cell surface antigen CD133 is expressed on early HSCs and EPCs, both of which collaborate to promote vascularization of ischemic tissues. 30 CD133 + cells can integrate into sites of neovascularization and differentiate into mature endothelial cells. Because CD133 expression is lost on myelomonocytic cells, this marker provides an effective means to distinguish “true” CD133 + EPCs from EPCs of myelomonocytic origin. 26 Less than 1% of nucleated BMCs are CD133 + , and because these cells cannot be expanded ex vivo, only limited numbers of CD133 + cells can be obtained for therapeutic purposes.

Mesenchymal Stem Cells

MSCs represent a rare population of CD34 − and CD133 − cells present in bone marrow stroma (10-fold less abundant than HSCs) and other mesenchymal tissues. 31 MSCs can readily differentiate into osteocytes, chondrocytes, and adipocytes. Differentiation of MSCs to cardiomyocyte-like cells has been observed under specific culture conditions and after injection into healthy or infarcted myocardium in animals. 32–34 When injected into infarct tissue, MSCs may enhance regional wall motion and prevent remodeling of the remote, noninfarcted myocardium. 34,35 Little is known about the effects of MSCs on myocardial perfusion. It is interesting to note however, that cultured MSCs secrete angiogenic cytokines, which improve collateral blood flow recovery in a murine hind limb ischemia model. 10 Because MSC clones can be expanded in vitro, and reportedly have a low immunogenicity, they might be used in an allogeneic setting in the future. 31

Skeletal Myoblasts

Skeletal myoblasts, or satellite cells, are progenitor cells that normally lie in a quiescent state under the basal membrane of mature muscular fibers. Myoblasts can be isolated from skeletal muscle biopsies and expanded in vitro. Myoblasts differentiate into myotubes and retain skeletal muscle properties when transplanted into an infarct scar. 36–39 Although myotubes do not couple with resident cardiomyocytes electromechanically, myoblast transplantation has been shown to augment systolic and diastolic performance in animal models of myocardial infarction. 40

Resident Cardiac Stem Cells

The presence of resident cardiac stem cell (CSC) population(s) capable of differentiating into cardiomyocyte or vascular lineages suggests that these cells could be used for cardiac tissue repair. 41–45 Intriguingly, CSCs can be clonally expand from human myocardial biopsies. 45 It has been reported that intramyocardial injection of these cells after AMI in mice promotes cardiomyocyte and vascular cell formation and leads to an improvement in systolic function. 45 If these findings can be reproduced, CSCs hold great promise for clinical applications, although it is conceivable that the bone marrow may contain a stem cell population with similar properties. 46

Embryonic Stem Cells

Embryonic stem (ES) cells are totipotent stem cells derived from the inner cell mass of blastocysts. Under specific culture conditions, ES cells differentiate into multicellular embryoid bodies containing differentiated cells from all three germ layers including cardiomyocytes. Human ES cell–derived cardiomyocytes display structural and functional properties of early-stage cardiomyocytes that couple electrically with host cardiomyocytes when transplanted into normal myocardium. 47,48 In theory, infinite numbers of cardiomyocytes could be obtained from human ES cell clones. However, unresolved ethical and legal issues, concerns about the tumorigenicity of the cells, and the need to use allogeneic cells for transplantation currently hamper their use in clinical studies. Eventually, nuclear transfer techniques may provide a means for generating an unlimited supply of histocompatible ES cells for the treatment of cardiac disease (therapeutic cloning). 49

Modes of Cell Delivery

The goal of any cell delivery strategy is to transplant sufficient numbers of cells into the myocardial region of interest and to achieve maximum retention of cells within that area. Retention may be defined as the fraction of transplanted cells retained in the myocardium for a short period of time (hours). The local milieu is an important determinant of cell retention, as it will influence short-term cell survival and, if a transvascular approach is used, cell adhesion, transmigration through the vascular wall, and tissue invasion.

Transvascular Approaches

Transvascular strategies are especially suited for the treatment of recently infarcted and reperfused myocardium when chemoattractants and cell adhesion molecules are highly expressed. 50–52

Intracoronary Artery Infusion

Selective intracoronary application delivers a maximum concentration of cells homogeneously to the site of injury during first passage. Unselected BMCs, circulating blood-derived progenitors cells, and MSCs have been delivered via the intracoronary route in patients with AMI and ischemic cardiomyopathy (Tables 1 and 3). In these studies, cells were delivered through the central lumen of an over-the-wire balloon catheter during transient balloon inflations to maximize the contact time of the cells with the microcirculation of the infarct-related artery. It is unknown whether this stop-flow technique is required to enhance cell retention within the infarcted area. In the hands of an experienced operator, intracoronary delivery is relatively easy to perform within less than an hour.

Table 1. Cell Therapy Trials in Patients With Acute Myocardial Infarction

Intravenous Infusion

In experimental models, intravenous delivery of EPCs or MSCs has been shown to improve cardiac function after AMI. 7,31,53 However, homing of cells to noncardiac organs limits the clinical applicability of this approach. 54,55 Indeed, in a recent study in post-AMI patients, significant myocardial homing of unselected BMCs was observed only after intracoronary stop-flow delivery but not after intravenous application. 56

Mobilization of Stem and Progenitor Cells

Considering that the acutely infarcted myocardium recruits circulating stem and progenitor cells to the site of injury, 7,53,57,58 stem and progenitor cell mobilization by cytokines may offer a noninvasive strategy for cardiac regeneration. This concept has been tested in animal models of AMI 59–63 and in pilot studies in patients with AMI and chronic myocardial ischemia. 64,65

Direct Injection in the Ventricular Wall

Direct injection is the preferred route for cell delivery in patients presenting late in the disease process when an occluded coronary artery precludes transvascular cell delivery (patients with chronic myocardial ischemia) or when cell homing signals are expressed at low levels in the heart (scar tissue). However, direct injection of cells into ischemic or scarred myocardium creates islands of cells with limited blood supply and may lead to poor cell survival. 66 Direct injection techniques are especially suited for the application of large cells, such as MSCs or myoblasts, which may cause microembolization after intracoronary delivery. Direct injection techniques have been used in patients with advanced coronary artery disease (Table 2) and in patients with ischemic cardiomyopathy (Table 3). Cell delivery by direct injection may be technically challenging in patients with AMI, particularly if cells are to be injected into the border zone of the infarct. The safety of such an approach remains to be established because perforation of the friable necrotic tissue remains a matter of concern.

Table 2. Cell Therapy Trials in Patients With Myocardial Ischemia and No Revascularization Option

Table 3. Cell Therapy Trials in Patients With Ischemic Cardiomyopathy

Transendocardial Injection

Using an injection needle catheter advanced across the aortic valve and positioned against the endocardial surface, cells can be directly injected into the left ventricular (LV) wall. 21–24,67 Electromechanical mapping of the endocardial surface can be used to delineate viable, ischemic, and scarred myocardium before cell injections. Average mapping and injection procedure times between 60 and 200 minutes have been reported. 21–24

Transepicardial Injection

Transepicardial cell injection has been performed as an adjunct to coronary artery bypass grafting (CABG). Transepicardial cell injection during open heart surgery allows for a direct visualization of the myocardium and a targeted application of cells to scarred areas and/or the border zone of an infarct scar. The invasiveness of this approach hampers its use as a stand-alone therapy. Conversely, the efficiency of cell transplantation may be difficult to evaluate and ascertain if CABG is performed simultaneously.

Transcoronary Vein Injection

A catheter system incorporating an ultrasound tip for guidance and an extendable needle for myocardial access has been used to deliver BMCs through the coronary veins into normal pig myocardium. 68 The same approach has been used in a pilot trial in patients with ischemic cardiomyopathy to deliver myoblasts to areas of nonviable myocardium. 69 In contrast to the transendocardial approach, where cells are injected perpendicular to the ventricular wall, the composite catheter system delivers cells parallel to the ventricular wall and deep into the injured myocardium. However, positioning of the injection catheter in a specific coronary vein is not trivial in all cases. 69

Clinical Applications of Stem Cell Therapy

Acute Myocardial Infarction

Modern reperfusion strategies and advances in pharmacological management have resulted in an increasing proportion of AMI survivors at heightened risk of developing adverse LV remodeling and heart failure. None of our current therapies addresses the underlying cause of the remodeling process, ie, the damage of cardiomyocytes and the vasculature in the infarcted area.

Experimental Background

In one of the earliest studies, HSCs were injected into the infarct border zone after coronary artery ligation in mice. Several days later, the infarcted area was replaced by newly formed myocardium with HSC-derived myocytes and vascular structures. 4 Transdifferentiation to cardiomyocytes and vascular structures has also been reported after transfer of CD34 + cells into mice with AMI. 58 Recent studies questioning that HSCs can transdifferentiate to cardiomyocytes when transplanted into infarcted murine myocardium have ignited a heated debate. 12,70,71 Yet, although data have been presented to support and to refute this idea, both sides agree that HSC transplantation can improve cardiac function after AMI. 4,12 Improvement of cardiac function has also been observed after transplantation of unselected BMCs or EPCs. Although myocyte formation did not occur, cells were shown to secrete angiogenic ligands, to incorporate into foci of neovascularization, and to improve regional capillarization and blood flow. 8,53,72

Clinical Trial Experience

Inspired by the exciting experimental data, several trials were initiated to test whether cell therapy is safe and feasible in patients after AMI. Some have decried the clinical trials as being premature without a more complete understanding of the underlying mechanisms, 71 whereas others have pointed out that the clinical trials are justified by the potential benefits of cell therapy. 73 All clinical studies included patients with AMI who had undergone primary angioplasty and stent implantation to reopen the infarct-related artery, and cells were infused intracoronarily by using the stop-flow balloon-catheter approach. In this regard, the clinical studies differ significantly from the animal studies, where the infarct-related artery was not reperfused and cells were directly injected into the myocardium. 4,8,12,53 The clinical trials may be categorized into studies using unselected BMCs or selected cell populations (Table 1).

Unselected Bone Marrow Cells

The combined experience from more than 100 patients suggests that intracoronary delivery of unselected BMCs (all nucleated cells or mononuclear cell fraction only) is safe in the short- and mid-term (several months). 13,14,16–19 No bleeding complications were noted after bone marrow harvest. Intracoronary BMC infusions did not appear to inflict additional ischemic damage to the myocardium or to promote a systemic inflammatory reaction, because no further increases in serum troponin or CRP levels were observed. No increased rates of in-stent restenosis were observed after transfer of unselected BMCs. 16,17,19 It should be mentioned that one patient developed in-stent thrombosis of the target vessel three days after cell infusion. Two days later, this patient also developed in-stent thrombosis in an unrelated coronary artery and went into fatal cardiogenic shock. Although this patient may have had an intrinsic tendency to develop in-stent thrombosis, it cannot be excluded that this complication was somehow related to cell therapy. 16 Clinical surveillance, Holter monitoring, and data from an electrophysiological study indicate that intracoronary BMC transfer is not associated with an increased propensity to ventricular (or supraventricular) arrhythmias. 13,14,16–19 Direct injection of filtered nucleated BMCs into the acutely infarcted myocardium in rats has been found to induce intramyocardial calcifications. 74 No evidence for intramyocardial calcifications (or tumor formation) has been obtained in patients 12 to 18 months after intracoronary delivery of Ficoll or gelatin gradient-purified BMCs. 16,75

Except for one study that included only five patients and no control group, 18 all trials indicate that intracoronary transfer of unselected BMCs enhances regional wall motion in the infarcted area. 13,14,17,19 In the three largest studies, this was associated with an increase also in global LVEF. 14,17,19 In contrast to earlier trials that included nonrandomized control groups, 13,14,17 the BOne marrOw transfer to enhance ST-elevation infarct regeneration (BOOST) trial included a randomized control group. 19 In the BOOST trial, BMC transfer resulted in an improvement of LVEF of six percentage points as compared with the control group after 6 months. For comparison, improvements of three to four percentage points are achieved by primary angioplasty and stent implantation in AMI and this results in better clinical outcomes as compared with thrombolytic strategies. 76,77 Improvement of LVEF was due mostly to improved regional wall motion in the infarct border zone. 19 Importantly, the effects of BMC transfer were observed on top of the benefits associated with established interventional and medical strategies to promote functional recovery after AMI. 19 In contrast to earlier nonrandomized studies, 13,14 a significant reduction of infarct size was not observed in the BOOST trial. 19 However, larger trials are required to further clarify the issue whether formation of new muscle tissue can be achieved by BMC transfer. So far, no trial has demonstrated a significant effect of BMC transfer on LV end-diastolic volumes, suggesting that unselected BMCs may have a limited impact on LV remodeling after AMI. 13,14,17–19 Again, larger studies are required to settle this issue. Follow-up data from the BOOST trial show that the improvement of LVEF is maintained after 18 months and indicate that BMC transfer prevents progression of diastolic dysfunction after AMI. 75,78

Selected Bone Marrow Cell Populations

The Transplantation Of Progenitor Cells And Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI) trial compared unselected mononuclear BMCs with circulating blood-derived progenitor cells (mostly EPCs). Both cell types appeared to have similar safety and efficacy profiles. 14,16

The therapeutic effects of MSC transplantation after AMI have been investigated in one clinical trial. 79 No arrhythmias or other side effects were observed. Unfortunately, it was not reported whether intracoronary MSC delivery promoted ischemic damage to the myocardium, 79 a complication that has occurred after intracoronary MSC infusions in dogs. 80 Six months after MSC-transfer, regional wall motion and global LVEF were improved and LV end-diastolic volume was decreased compared with a randomized control group that had received an intracoronary infusion of saline. 79

In another clinical trial using selected BMC populations, CD133 + cells were infused into the infarct-related artery. 81,82 After 4 months, 6 of 14 patients had developed a significant in-stent restenosis or complete reocclusion, and two had developed a de novo lesion in the infarct-related artery. 82 These numbers are worrisome, but the study may be too small to establish that these side effects are causally related to CD133 + cell transfer. Global LVEF, regional wall motion, and tissue perfusion increased in the cell transfer group but not in a cohort of matched control patients. 81 However, firm conclusions regarding efficacy cannot be derived from this small pilot trial.

Stem and Progenitor Cell Mobilization

Stem cell mobilization with stem cell factor (SCF) and/or granulocyte colony-stimulating factor (G-CSF) has been proposed to stimulate myogenesis and angiogenesis in the infarcted area and to improve cardiac function after AMI in mice. 59,60 By contrast, treatment with SCF and G-CSF enhances vascularization of the infarcted area but does not improve cardiac function in baboons after AMI. 63 Perhaps, reperfusion of the infarct-related artery before cytokine therapy would have permitted better access of mobilized cells to the infarct center in this large animal model. 63 Of note, G-CSF may accelerate infarct healing by enhancing macrophage infiltration and matrix metalloproteinase activation 62 and suppress cardiomyocyte apoptosis by activating the cytoprotective STAT3 transcription factor, 83 suggesting that stem cell–independent mechanisms may contribute to the effects of G-CSF after AMI.

In a first clinical investigation, 10 patients presenting with myocardial infarction 2 to 270 days after symptom onset were treated with G-CSF at 10 μg/kg body weight for 4 days. Patients then underwent angioplasty and stent implantation of the infarct-related artery. In seven of these patients, G-CSF mobilized peripheral blood-derived leukocytes were collected just before the intervention and infused into the infarct-related artery after stent placement. No deaths, substantial arrhythmias, aggravation of heart failure, or angina occurred during G-CSF administration and a 6-month follow-up period. However, cell infusions resulted in a 65% increase in serum creatine kinase-MB levels, indicative of mild myocardial damage. More seriously, 7 of the 10 patients developed in-stent restenosis at 6 months, which prompted a premature termination of the study. 84 It should be pointed out that vascular injury by balloon angioplasty and stenting had been performed in this study while systemic leukocyte counts were greatly elevated. G-CSF has the potential to activate neutrophils, for example by stimulating adhesion to endothelial cells thereby influencing their recruitment at sites of inflammation and tissue injury. 85 These systemic effects of G-CSF may have contributed to excess neointima proliferation and restenosis. Although an improvement in LVEF was observed in patients receiving G-CSF and cell infusions, 84 interpretation of this finding is impossible without a control group.

In a more recent study, 15 patients with AMI were treated with G-CSF at 10 μg/kg body weight for 6 days, starting 80±30 minutes after primary angioplasty and stent implantation of the infarct-related artery. 64,86 G-CSF treatment after stent implantation was not associated with an enhanced rate of in-stent restenosis, or other serious adverse events. 86 Compared with a randomized control group, patients receiving G-CSF experienced a more pronounced recovery of global LVEF after 4 months. However, the beneficial effects of G-CSF were magnified by an unexpected decrease in LVEF in the control group. 64

Directions for Future Clinical Research

Brief intermittent periods of ischemia applied at the onset of reperfusion reduce infarct sizes in animal models of myocardial ischemia and reperfusion (ischemic postconditioning). 87 In the cell therapy trials, stop-flow cell delivery was performed several days after reperfusion, making postconditioning an unlikely explanation for the effects of cell therapy. Nevertheless, future trials need to include control groups that undergo bone marrow harvest and an intracoronary sham infusion to unambiguously establish that cell transfer, and not bone marrow puncture or intracoronary manipulation, mediates the functional improvements.

Therapeutic effects of selected versus unselected BMC populations should be compared head-to-head. Assuming that unselected BMCs contain effective and ineffective cell populations, and that both may be recruited to the infarcted area by similar mechanisms, 52 purification and infusion of the effective cell population(s) only may allow more of the effective cells to transmigrate into the infarcted area.

Similar benefits have been reported after delivery of greatly variable numbers of mononuclear BMCs. 13,14,17 Along this line, the absolute numbers of transplanted nucleated cells, CD34 + cells, and colony-forming stem cells did not correlate with subsequent improvements in LVEF in the TOPCARE-AMI and BOOST trials. This may be because the cell numbers infused were within a narrow range, or because differences in the functional capacity of the cells may override differences in cell numbers. 15,19 Intriguingly, labeling studies indicate that less than 3% of unselected BMCs are retained in the infarcted area after intracoronary delivery in patients. 56 Although this rate of retention was sufficient to improve LV systolic function in the BOOST trial, 19 dose-finding studies are required to define the optimum cell number.

Post hoc analyses of the BOOST trial database suggest that the effects of BMC transfer are consistent across several subgroups defined according to sex, age, infarct size and territory, time from symptom onset to reperfusion, and baseline LVEF. 19 Specific subgroups need to be defined prospectively in future trials, eg, patients presenting late after symptom onset in whom little myocardial salvage can be expected from reperfusion therapy. 88 For safety concerns, patients with AMI and heart failure symptoms have been excluded from previous trials. Considering that these patients may benefit most from an improvement in LVEF, future trials should assess the effects of cell therapy in this patient subgroup.

G-CSF and other cytokines with stem-cell mobilizing and cardioprotective properties should be further evaluated as stand-alone therapy or in combination with cell transfer. 89,90 Depending on whether cytokine and stem cell therapy improve cardiac function via shared or distinct mechanisms, cytokines might evolve as a noninvasive alternative or as an adjunct to cell therapy.

Ultimately, a large outcome trial looking at a combined mortality and morbidity end point will have to be conducted. One way to limit the size of such a trial would be to include only patients with AMI complicated by severe LV systolic dysfunction or heart failure symptoms, ie, patients who carry a high risk of future adverse events. Still, thousands of patients will have to be randomized to demonstrate an added benefit of cell therapy on clinical outcomes on top of our current, interventional, and medical therapies.

Coronary Artery Disease With No Mechanical Revascularization Option

Despite significant advances in coronary revascularization techniques, some patients with coronary artery disease and myocardial ischemia have no revascularization option because of the diffuse nature of their disease. A number of these patients experience anginal symptoms despite maximal medical therapy. Chronic myocardial ischemia can be associated with a regional impairment of contractile function, which is partially reversible when tissue perfusion is restored (hibernating myocardium). Moreover, ischemia increases the risk of arrhythmias and sudden cardiac death. There is a clear need for new therapeutic strategies aimed at delivering oxygenated blood to the myocardium in these patients.

Experimental Background

Transendocardial injection of unselected BMCs or EPCs enhances collateral flow, capillary density, and regional contractility in pigs with chronic myocardial ischemia 9,72 The mechanisms how BMC injections enhance myocardial perfusion are unknown. Bone marrow–derived EPCs have been proposed to enhance tissue perfusion by differentiating into endothelial cells at sites of neovascularization. 7,91,92 Recent articles have highlighted the potential of BMCs to deliver a natural cocktail of angiogenic and arteriogenic cytokines to the myocardium. 9,10,22,93 In that regard, cell therapy may have advantages above previous single-cytokine gene therapy approaches to treat patients with chronic myocardial ischemia. It has also been reported that regional perfusion and contractile function of hibernating pig myocardium can be improved by G-CSF, suggesting that stem and (endothelial) progenitor cell mobilization may represent an alternative, less invasive therapeutic strategy. 94

Clinical Trial Experience

Unselected mononuclear BMCs have been used in several small studies in patients with coronary artery disease not amenable to conventional revascularization techniques (Table 2). 20–24 In a first study, five patients undergoing CABG received transepicardial BMC injections into an ischemic area with no graftable vessel. All patients had an uneventful postoperative course. No arrhythmias occurred, and no intramyocardial calcification or tumor formation was observed after 1 year, suggesting that the procedure may be safe. 20 Myocardial perfusion in the injected area improved in three of these patients. 20 In three additional studies, mononuclear BMCs were injected transendocardially into ischemic myocardium under electromechanical guidance. 21–24 No procedure-related complications were reported, and no sustained ventricular arrhythmias were observed up to 1 year after cell transfer. 21–24 One patient died suddenly 14 weeks after cell transfer. 23 Although sudden (cardiac) death is a typical complication in patients with severe ischemic heart disease, it cannot be ruled out that this death was related to cell injections. Improvements of anginal symptoms, exercise capacity, regional tissue perfusion, and LV systolic function have been reported after intramyocardial BMC injections (Table 2).

A recent study investigated the effects of G-CSF on symptoms and myocardial perfusion in 16 patients with intractable angina. 65 Treatment with G-CSF (10 μg/kg body weight for 5 days) promoted a strong increase in circulating EPC numbers and an improvement in anginal symptoms. However, there was no objective evidence of enhanced myocardial perfusion or improved regional wall motion. Furthermore, two patients experienced myocardial infarctions, raising concerns about the safety of G-CSF in this patient population. 65

Directions for Future Clinical Research

Intramyocardial injection of unselected BMCs is feasible and appears to be safe in patients with chronic myocardial ischemia. 21–24 The efficacy of this approach is unknown because none of the previous trials included a randomized control group. Our experience with transmyocardial laser revascularization has highlighted the need for control groups undergoing sham-catheterization and intramyocardial sham-injections to control for the placebo effect typically observed in this patient population. 95 Still, the idea to improve myocardial perfusion by BMC injections is intriguing and should be tested prospectively in larger, randomized clinical trials. Because symptomatic improvement is the major goal of cell therapy in this patient population, it will be very important to establish the safety of procedure.

Although no adverse coronary events have been observed after short-term administration of G-CSF to normal volunteers, 96 G-CSF may not be safe in patients with advanced coronary artery disease. 65 Any future trial investigating the role of G-CSF in these patients should use a careful dose-escalating regime. Alternative strategies to promote EPC mobilization and, possibly, angiogenesis in ischemic myocardium should be explored (eg, statins, cytokines, physical exercise). However, a limitation of any stem cell mobilizing strategy may be that circulating cells have insufficient access to severely ischemic myocardium.

Ischemic Cardiomyopathy, Chronic Heart Failure

Chronic heart failure has emerged as a major worldwide epidemic. Recently, a fundamental shift in the underlying etiology of heart failure is becoming evident, in which the most common cause of heart failure is no longer hypertension or valvular disease, but rather long-term survival after AMI. Conceptually, replacement of akinetic scar tissue by viable myocardium should improve cardiac function and impede progressive LV remodeling.

Experimental Background

Among various indications for stem cell therapy that can be envisioned, repair of scar tissue is the most challenging. Transplanted cells will face limited blood supply and may not receive the environmental cues essential for transdifferentiation into vascular cells (or cardiomyocytes). Transplantation of myoblasts, which supposedly have a good tolerance to ischemia and are committed to differentiate along the myocyte lineage, may therefore be a valuable option in this setting. Indeed, injection of myoblasts into infarcted myocardium has been shown to improve LVEF and to ameliorate adverse LV remodeling in small and large animal species. 36,97,98 Although grafted myotubes may contract in response to electrical stimulation, 37,38 they do not express the intercalated disk proteins N-cadherin or connexin 43, indicating that they are not electromechanically coupled to their host cardiomyocytes. 99 Therefore, improvement of cardiac function observed in animal models after myoblast transplantation may not depend on synchronized contractile activity of the cells. In line with this hypothesis, it has been shown that the functional benefits of myoblast transplantation in a rat infarct model are sustained over time despite a progressive loss of engrafted cells. 100 Injected myoblasts do not appear to stimulate angiogenesis locally. 101 However, it has been hypothesized that myoblasts may release paracrine factors instructing neighboring cardiomyocytes to maintain their replicative potential or to favor differentiation of CSCs into cardiomyocytes. 38 Transplanted myoblasts can fuse with cardiomyocytes at the graft-host interface. Although fusion appears to be a rare event, the functional properties of these hybrid cells need to be further explored. 102

Recent studies have investigated whether injection of BMCs can be used to regenerate recently infarcted myocardium. Injection of CD133 + stem cells into the infarcted myocardium 10 days after coronary artery ligation in rats promoted an increase in LVEF. 101 By contrast, direct injection of unselected BMCs 3 weeks after coronary ligation in sheep did not enhance functional recovery, 66 emphasizing that the cell type and cell delivery method need to be carefully adapted to the underlying disease state.

Clinical Trial Experience

Skeletal Myoblasts

After an initial case report, 103 several small trials investigating the safety and feasibility of myoblast transplantation in patients with ischemic cardiomyopathy have been published (Table 3). These studies indicate that it is feasible to establish and expand myoblast cultures from skeletal muscle biopsies and to obtain target myoblast numbers within 2 to 3 weeks. 39,67,104–107 One major safety concern has arisen from these studies, ie, that myoblast grafts may represent an arrhythmogenic substrate. 108 In the first clinical trial, 104 10 patients with severely reduced LVEF undergoing CABG received myoblast injections into scar tissue supplied by a totally occluded, nongraftable coronary artery. In four of these patients, sustained ventricular tachycardias occurred between 11 and 22 days after surgery. Two of these patients had additional episodes of ventricular tachycardias 5 and 9 months after the operation. All four patients were treated with an implantable cardioverter-defibrillator (ICD). 104 In two similar studies, 21 additional patients received myoblast injections. In these studies, surgical revascularization involved the noninjected and the injected areas. 105,106 Two patients developed ventricular tachycardias 1 day after surgery 106 and one patient on day 40. 105 In another report, myoblasts were injected into scarred myocardium in five patients with end-stage ischemic heart failure undergoing LV assist device implantation as a bridge to heart transplantation. Two of these patients had ventricular tachycardias in the immediate postoperative period, one of whom already had a history of ventricular arrhythmias. 39 Finally, myoblasts have been injected transendocardially as a stand-alone procedure in five patients with heart failure after an anterior wall AMI. 67 In one patient, a minor elevation of serum troponin levels was noted after the procedure. More seriously, another patient had ventricular tachycardias 6 weeks after myoblast injections and underwent ICD implantation. 67 Two sudden deaths and three ventricular arrhythmias have occurred in eight additional patients treated by transendocardial myoblast injections. 67 Episodes of ventricular tachycardias were also observed in one of nine patients with postinfarction heart failure receiving transcoronary vein injections of myoblasts. 69 It is likely, therefore, that myoblast injections increase the risk of ventricular arrhythmias in this patient population. 109 In the absence of electromechanical coupling, the underlying mechanisms remain uncertain. It has been proposed that the ability of myoblasts to fire bursts of action potentials may induce deleterious extrasystoles, even in the absence of electromechanical coupling, through electrotonic interaction. 38,108 Moreover, local tissue injury could be responsible for arrhythmogenesis. 104,108 In a recent study, no serious arrhythmias were observed in 20 patients during a mean followup of 14 months after injecting myoblasts that had been expanded in autologous rather than fetal bovine serum, 107 leading to the hypothesis that trace contamination with xenogenic proteins may provoke an arrhythmogenic immune reaction at the injection site. 107

In most trials, improvements of regional wall motion and global LVEF have been noted after myoblast injections. 67,104–107 Moreover, evidence for enhanced viability in the injected myocardial areas has been obtained in some of these reports. 105,107 However, because of the small number of patients, lack of control groups, and the confounding effect of concomitant revascularization, no firm conclusions regarding efficacy can be drawn at this time.

Bone Marrow Cells

In one trial, CD133 + BMCs were injected transepicardially into the infarct border zone in 12 patients undergoing CABG of noninjected myocardial areas. 110,111 In contrast to the myoblast trials, infarcts had occurred fairly recently in the patients in this study (Table 3). No procedure-related complications were reported, and no serious ventricular arrhythmias were observed up to 14 months. 111 After 6 to 8 months, perfusion of the cell-injected area and LVEF were improved. However, because there was no control group, the efficacy of this approach remains uncertain.

In a recent trial, 86 patients with ischemic cardiomyopathy received intracoronary infusions of unselected mononuclear BMCs or of circulating blood-derived progenitor cells by the stop-flow balloon catheter technique. The procedure was safe. 112 After 3 months, LVEF in the BMC group was improved by three percentage points, but did not change significantly in control patients and in the progenitor cell group. 112

Directions for Future Clinical Research

Randomized, double-blind trials are needed to rigorously evaluate the safety and efficacy of cell therapy in patients with ischemic heart failure. It may be advisable to restrict the use of myoblast transplantation to patients with an ICD. The monitoring function of the ICD will provide critical information on the natural course of myoblast-induced arrhythmias. 108 If the implantation of an ICD will be routinely required in patients receiving myoblast injections, the procedure might not be cost-effective.

Postmortem studies indicate that only a small fraction of injected myoblasts survive in scarred human myocardium. 39,113 Accordingly, preimplantation antiapoptotic treatments or coinjection of angiogenic growth factors may enhance myoblast survival after transplantation. 109,114 Another future strategy may involve the ectopic expression of connexin 43 in myoblasts, which may permit electrical coupling with resident cardiomyocytes. 115

It is interesting to note that intracoronary infusions of mononuclear BMCs or blood-derived progenitor cells promoted greater improvements of LVEF in patients with AMI as compared with patients with ischemic cardiomyopathy. 14,112 Because cell retention may be limited after intracoronary delivery into chronically infarcted myocardium, pharmacological or genetic approaches to enhance cell retention and engraftment should be explored.

Considering that functional benefits of cell transplantation have also been observed in animals with dilated cardiomyopathy, 116 future trials may want to explore the role of cell therapy in patients with nonischemic heart failure. In this regard, a pilot study suggests that intracoronary BMC-transfer may be safe and potentially effective in patients with Chagas cardiomyopathy. 117

Issues to Be Addressed in Future Studies

So far, a flurry of small, mostly uncontrolled clinical studies exploring the safety and feasibility of stem cell therapy have been conducted. These studies have used a myriad of different cell types and preparations, each in a small number of patients with different disease states. In the aggregate, this preliminary clinical evidence suggests that stem cell therapy might work. Although these initial clinical studies have generated a great deal of hope, we should take into account the lessons learned from the translation of therapeutic angiogenesis into clinical studies, where great expectations raised by open studies have not been confirmed by subsequent randomized trials. We advocate to no longer perform studies involving small numbers of patients, but rather to conduct intermediate-size, double-blind, randomized-controlled clinical trials to establish the effects of stem cell therapy on surrogate markers, like LVEF, myocardial perfusion, or exercise capacity. Upcoming trials should also address procedural issues such as the optimal cell type, cell dosage, and timing of cell transfer. These trials may also look at combined morbidity and mortality end points, although they may be too small to be conclusive in this regard. Safety remains the key concern as we proceed.

Although these studies are underway, fundamental questions need to be addressed experimentally. What is the fate of the injected cells after transplantation? How long do they survive? Do the cells incorporate, or is transient retention sufficient to promote functional effects? Genetic and transgenic markers should be used to determine the lineage commitment of engrafted cells. We would encourage laboratories that have arrived at discrepant conclusions regarding cell fate and lineage commitment to share their experimental protocols. Cell labeling and imaging techniques need to be developed to track stem cell fate in patients and correlate cell retention and engraftment with functional outcomes. Emerging evidence suggests that transplanted stem cells may interact with resident CSCs to enhance their regenerative potential. What is the nature and functional relevance of this interaction? Can CSCs be used for cardiac repair, or is their potential similar to cells obtained from bone marrow? Can the regenerative capacity of transplanted stem cells be enhanced by drugs, cytokines, or gene therapy approaches? Pharmacological and genetic strategies may help to enhance stem cell retention, engraftment, differentiation, and paracrine capability. 34,118–120

In the era of evidenced-based medicine, effects on surrogate markers will not suffice to establish stem cell therapy as a valid therapeutic option for patients with cardiovascular disease. Outcome trials will have to be conducted. Funding is an issue, unless intellectual property is involved or public programs are being developed. Support from governmental organizations or charities will be required to ensure that cell therapies, which may be efficacious but commercially less attractive (eg, unselected BMCs), will undergo much-needed further clinical testing. Support may also come from device manufactures, if a specific cell therapy approach relies on an optimized cell delivery system, or if a device is developed that allows cardiologists to obtain cell preparations “at the bedside” without the requirement for a local GMP facility. It can be anticipated that cell preparation and delivery devices will undergo considerable development once the clinical benefit of cell therapy is clearly established. Some companies have already started to develop and market cell therapy products, for example, culture-expanded autologous skeletal myoblasts for use in patients with ischemic cardiomyopathy, or cryopreserved allogeneic MSCs as an “off-the-shelf” therapeutic for patients with AMI.

Original received November 19, 2004 revision received December 22, 2004 accepted December 27, 2004.


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