Adult Stem Cells

Adult stem cells (ASCs) represent ethically acceptable source of stem cells and are eligible for federal funding in the USA and elsewhere ( Fig. 25.2 ). ASCs reside within different tissues as tissue-specific progenitors that are employed during tissue self-renewal and/or differentiation. When transplanted into the host, ASCs exhibit lower tumorigenic potential than hESCs. ASCs play an important role in the lifelong maintenance of tissue homeostasis . Any homeostatic disturbance can disrupt a dynamic balance between self-renewal and differentiation which can lead to excessive differentiation, resulting in the exhaustion of the stem cell pool or overproduction resulting in tumorigenesis .

Bone marrow represents a typical adult stem cell source, containing different cell populations that have the potential to migrate and transdifferentiate into cells of diverse phenotypes . The plasticity of bone marrow-derived stem cells has been broadly studied. Two major subsets of bone marrow cells include hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). HSCs are commonly identified by the expression of CD34 ⁺ cell surface antigen; HSCs have been studied extensively and used clinically for bone marrow transplantation in a variety of hematological disorders .

MSCs are a population of cells present in adult tissues including the bone marrow and adipose tissues . They have been shown to differentiate into multiple tissues including neuronal, adipose, muscle, liver, lungs, spleen and gut. MSCs derived from adipose tissue are particularly attractive because they are easily accessed and not in short supply.

In summary, ASCs represent an ethically acceptable source of stem cells, albeit currently of limited therapeutic use except for HSCs. The major reasons are the challenges involved in isolating scarce ASCs, their restricted differentiation potential, and difficult and often unsuccessful ex vivo expansion . If these drawbacks were successfully resolved, ASCs could become an important source of tissue-specific stem cells for regenerative therapies.

Multipotent Stem Cells from Extraembryonic Tissues

Reprogramming of Somatic Cells: Induced Pluripotent Stem Cells

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells ( Fig. 25.2 ) by the addition of several genes, as initially reported by Yamanaka’s and Thomson’s laboratories . Yamanaka and colleagues generated human iPS cells through retrovirus-mediated transfection of the genes POU5F1 , SOX2 , KLF4 and c-MYC , while Thomson and colleagues generated iPS cells without the oncogene c-MYC , inducing the pluripotent state by retroviral transfection of POU5F1 , SOX2 and NANOG . It was demonstrated that iPS cells are similar to hESCs by morphology, proliferative capacity and pluripotency. Since these initial reports on iPS cell derivation, a variety of different methods has been reported for reprogramming (see Chapter 13 ) .

Reprogramming is an exciting approach as it does not require the use of human oocytes and/or embryos. Induced pluripotent stem cells opened new possibilities particularly for interrogating human disease. Derivation of patient-specific iPS cells will recapitulate disease pathology and this type of research will give important insight into the pathogenesis of many degenerative diseases. Also, iPS cells are an extensive source of histocompatible pluripotent stem cells, which makes them particularly appealing for clinical use. However, there are several important concerns to be resolved: the low efficiency of reprogramming, tumor formation, and developmental equivalence between iPS cells and hESCs. Two recent reports show that reprogrammed cells fail to differentiate or do not differentiate as efficiently as hESCs . These findings clearly demonstrate that iPS cells are distinct population of pluripotent stem cells and indicate the imperfections in the reprogramming process. Further research is needed to diagnose and overcome these deficiencies of iPS cells prior to their use in regenerative medicine.

Altered Nuclear Transfer

Altered nuclear transfer (ANT) is a theoretical method for derivation of pluripotent stem cells that would avoid embryo destruction . It is based on somatic cell nuclear transfer (SCNT), with a modified nucleus that would lead to an embryo-like structure incapable of implantation ( Fig. 25.2 ). It has been shown in mice that embryos with a mutation in the Cdx2 gene die at the blastocyst stage since trophectoderm fails to form, while the ability to derive hESCs from the inner cell mass was unchanged . It remains unknown whether CDX2 is the optimal target in human development and whether such embryos could be used to derive stem cells . It is also unclear whether these implantation-disabled structures could be considered as biological artifacts and not as defective embryos . Finally, the requirement for a large supply of human oocytes, if ANT should prove successful, implicates further ethical challenges .

Single Blastomere Biopsy

Lanza and co-workers developed single blastomere biopsy (SBB) for the purpose of deriving hESCs in a manner that would avoid embryo destruction . This approach uses a technique that was originally developed for preimplantation genetic diagnosis (PGD) , the difference being the derivation of hESCs from a single removed blastomere rather than genetic testing . A biopsied embryo can continue developing and reach the blastocyst stage and beyond, which was demonstrated with more than a decade of experience with PGD . This method has been used successfully to derive hESC lines . Although this approach bypasses the ethical issue of embryo destruction it brings the risk associated with embryo biopsy which is accepted as the part of PGD procedure, but it would be considered unjustified in the absence of a clinical indication . In addition, US regulations forbid research on an embryo that imposes greater than minimal risk, unless the research is for the direct benefit of the fetus . To date, none of the hESC lines derived by SBB has been approved for National Institutes of Health (NIH) funding .

Organismically Dead Embryos

Embryos engendered by in vitro fertilization (IVF) procedures frequently fail to develop properly and are classified as non-viable . Many non-viable embryos have undergone an irreversible arrest of normal development. Aneuploidy is widespread but mosaicism is common with normal cells distributed among the abnormal. Even a few- celled embryo is an organism with cell-cell communication essential for normal growth and development. Normal cells deprived of integration fail to develop and die. Irreversibly arrested, non-viable embryos satisfy the universal concept of death for multicellular life: an irreversible loss of integrated organismic function. We proposed that hESCs could be derived from cells harvested from human embryos that have died, despite best efforts, during the course of IVF for reproductive purposes . This approach, wherein live cells are harvested from dead embryos is analagous to the harvesting of essential organs from deceased donors and the established ethics of essential organ donation could be applied to the derivation of new hESC lines . The use of brain death as a criterion for diagnosing the death of a developed human informs the use of irreversible arrest of normal development in diagnosing the death of a developing human. The utility of a crisp diagnosis of death in the developed human avoids the inappropriate application of scarce medical resources and is the foundation of essential organ transplantation. Similar benefits would accrue to the diagnosis of death in the developing human.

The natural history studies that provided the foundation for diagnostic criteria for brain death inspired similar studies of non-viable embryos. Observation of non-viable embryos that arrest and then decompose could provide a stage of development and a duration of arrest that define a time beyond which, having failed to develop, no development is possible. In a retrospective study of non-viable embryos, we determined that many of the non-viable embryos rejected for clinical use were organismically dead: the failure of normal cell division for 48 h was irreversible and, despite the possible presence of individual living cells, indicated an irreversible loss of integrated organismic function . A follow-up prospective study examined the progression of non-viable embryos in extended culture . The analysis showed that developmental arrest observed in some human embryos by embryonic day 6 (ED6) following IVF cannot be reversed by extended culture in conditions suitable for preimplantation embryos, as no morphological changes indicative of normal developmental progression were seen and no unequivocal instances of further cell divisions were observed . These observations are in line with standard IVF practice; such embryos are not transferred or cryopreserved because they are known not to produce live offspring . Although morphological categorization of embryos is of limited value in predicting cell number, the higher cell number associated with cavitation may predict greater potential for the success of hESC derivation . Also, the majority of irreversibly arrested embryos contain a high proportion of vital cells regardless of the stage of arrest, indicating that harvesting cells and deriving hESCs from such non-viable embryos should be feasible .

To date, 14 hESC lines have been successfully derived from non-viable embryos that were irreversibly arrested, according to these criteria, thus demonstrating the feasibility of this alternative approach . The first cell line (hES-NCL9) was derived from 132 arrested embryos . In a different study, 11 hESC lines were derived from 413 poor-quality embryos rejected for clinical use . Recently, our group has derived two hESC lines: CU1 and CU2 from 159 ED6 irreversibly arrested, non-viable human embryos . Pluripotency and differentiation potential were demonstrated in vitro and/or in vivo for all 14 hESC lines derived . While it is common for arrested embryos to be aneuploid , analysis revealed normal or apparently normal karyotype in all 14 hESC lines derived to date .

Derivation of hESC lines from organismically dead embryos has the likelihood of being accepted by the strongest opponents of hESC research . This approach is unique because it defines a common ground in the stem cell debate. Eleven hESC lines derived by Daley and colleagues have been included in the NIH stem cell registry and are available for research with NIH funding . Despite the low efficiency of isolation of hESCs from organismically dead embryos, large-scale derivation is feasible because in the US alone nearly half a million such embryos are generated yearly as a by-product of IVF for reproductive purposes . Available data suggest that hESCs derived from organismically dead embryos are of equal quality to lines derived by the classical, inner cell mass (ICM) derivation approach, but further characterization of these lines is needed .