Pluripotent stem cells derived from Organismically Dead Embryos (Landry-Zucker proposal).
Council preliminary evaluations in 2005 : Not yet tested, even in animals. Natural history studies proposed could be undertaken forthwith and in an ethical manner. "Ethically acceptable for basic investigation in humans"
Update : This approach was recently demonstrated for "arrested" human embryos. Zhang, X., P. Stojkovic, et al., Derivation of human embryonic stem cells from developing and arrested embryos, Stem Cells, 24: 2660-76 (2006).
Abstract : Human embryonic stem cells (hESC) hold huge promise in modern regenerative medicine, drug discovery, and as a model for studying early human development. However, usage of embryos and derivation of hESC for research and potential medical application has resulted in polarised ethical debates since the process involves destruction of viable developing human embryos. Here we describe that not only developing embryos (morulae and blastocysts) of both good and poor quality but also arrested embryos could be used for the derivation of hESC. Analysis of arrested embryos demonstrated that these embryos express pluripotency marker genes such OCT4, NANOG and REX1. Derived hESC lines also expressed specific pluripotency markers (TRA-1-60, TRA-1-81, SSEA4, alkaline phosphatase, OCT4, NANOG, TERT and REX1) and differentiated under in vitro and in vivo conditions into derivates of all three germ layers. All the new lines including line derived from late arrested embryo have normal karyotype. These results demonstrate that arrested embryos are additional valuable resources to surplus and donated developing embryos and should be used to study early human development or derive pluripotent hESC.
Pluripotent Stem Cells via Blastomere Extraction from Living Embryos
Council preliminary evaluations in 2005 : Now technically feasible, though large ethical difficulties remain. Ethically unacceptable in humans.
Update : It was shown that hESC cultures could be derived from single blastomeres, although in these experiments, all the human embryos were destroyed. Klimanskya, I. , Y. Chung, S. Becker, et al., Human embryonic stem cell lines derived from single blastomeres, Nature, Advance online publication, August 23, 2006.
Abstract : The derivation of human embryonic stem (hES) cells currently requires the destruction of ex utero embryos. A previous study in mice indicates that it might be possible to generate embryonic stem (ES) cells using a single-cell biopsy similar to that used in preimplantation genetic diagnosis (PGD), which does not interfere with the embryo's developmental potential. By growing the single blastomere overnight, the resulting cells could be used for both genetic testing and stem cell derivation without affecting the clinical outcome of the procedure. Here we report a series of ten separate experiments demonstrating that hES cells can be derived from single blastomeres. Nineteen ES-cell-like outgrowths and two stable hES cell lines were obtained. The latter hES cell lines maintained undifferentiated proliferation for more than eight months, and showed normal karyotype and expression of markers of pluripotency, including Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, nanog and alkaline phosphatase. These cells retained the potential to form derivatives of all three embryonic germ layers both in vitro and in teratomas. The ability to create new stem cell lines and therapies without destroying embryos would address the ethical concerns of many, and allow the generation of matched tissue for children and siblings born from transferred PGD embryos.
Pluripotent Stem Cells derived from Biological Artifacts
Council preliminary evaluations in 2005 : Technically the most demanding, and ethically the most complex and puzzling. It would need to be carefully tested in animals before any thought of human trials could be countenanced. Not at this time ethically acceptable for trials with human material.
Update : One possible approach appears to work in a first test in the mouse .
Meissner, A. and R. Jaenisch, Generation of nuclear transfer-derived pluripotent ES cells from cloned Cdx2-deficient blastocyst, Nature, 239: 212-5 (2006)
Abstract : The derivation of embryonic stem (ES) cells by nuclear transfer holds great promise for research and therapy but involves the destruction of cloned human blastocysts. Proof of principle experiments have shown that 'customized' ES cells derived by nuclear transfer (NT-ESCs) can be used to correct immunodeficiency in mice. Importantly, the feasibility of the approach has been demonstrated recently in humans, bringing the clinical application of NT-ESCs within reach. Altered nuclear transfer (ANT) has been proposed as a variation of nuclear transfer because it would create abnormal nuclear transfer blastocysts that are inherently unable to implant into the uterus but would be capable of generating customized ES cells. To assess the experimental validity of this concept we have used nuclear transfer to derive mouse blastocysts from donor fibroblasts that carried a short hairpin RNA construct targeting Cdx2. Cloned blastocysts were morphologically abnormal, lacked functional trophoblast and failed to implant into the uterus. However, they efficiently generated pluripotent embryonic stem cells when explanted into culture.
Pluripotent Stem Cells via Somatic Cell Dedifferentiation
Council preliminary evaluations in 2005 : ethically the most unobjectionable, but for now scientifically and technologically uncertain. Ethically unproblematic and acceptable for use in humans.
Update : Adding four genetic factors to mouse fibroblasts changes them into pluripotent stem cells. Takahashi, K. and S. Yamanaka, Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors , Cell, 126: 1-14 (2006) [Note that what was produced in these experiments were "genetically engineered" mouse pluripotent stem cells, which may or may not be the functional equivalent of mouse ESCs isolated from the inner cell mass of early mouse embryos.]
Abstract : Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. Here, we demonstrate induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions. Unexpectedly, Nanog was dispensable. These cells, which we designated iPS (induced pluripotent stem) cells, exhibit the morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent stem cells can be directly generated from fibroblast cultures by the addition of only a few defined factors.
Initial work with human materials using cell fusion rather than genetic factors:
Cowan, C.A. , et al., Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells , Science, 309: 1369-1373 (2005)
Non-embryo sources of "embryonic like", multipotent human stem cells
From human umbilical cord blood.
Council preliminary evaluations in 2005 : Not completely evaluated, although reference was made to this possibility on page 28
" .genetically matched stem cells can be more effectively derived using the newborn's umbilical cord blood (a well-established procedure), though it is unclear whether the stem cells isolatable from cord blood will have the same capabilities as embryonic stem cells",
and on page 52 of Alternative Sources of Human Pluripotent Stem Cells.
".further research - using also stem cells from umbilical cord blood - might very well turn out to yield the big payoff: fully pluripotent stem cells, obtainable at will and altogether without any involvement of embryos - and well suited for autologous transplantation".
Update : Zhao, Y., et al., Identification of stem cells from human umbilical cord blood with embryonic and hematopoietic characteristics, Experimental Cell Research, 312: 2454-2464 (2006)
Abstract : We identified stem cells from the umbilical cord blood, designated cord blood-stem cells (CB-SC). CB-SC displayed important embryonic stem (ES) cell characteristics including expression of ES-cell-specific molecular markers including transcription factors OCT-4 and Nanog, along with stage-specific embryonic antigen (SSEA)-3 and SSEA-4. CB-SC also expressed hematopoietic cell antigens including CD9, CD45 and CD117, but were negative for CD34. CB-SC displayed very low immunogenicity as indicated by expression of a very low level of major histocompatibility complex (MHC) antigens and failure to stimulate the proliferation of allogeneic lymphocytes. CB-SC could give rise to cells with endothelial-like and neuronal-like characteristics in vitro, as demonstrated by expression of lineage-associated markers. Notably, CB-SC could be stimulated to differentiate into functional insulin-producing cells in vivo and eliminated hyperglycemia after transplantation into a streptozotocin-induced diabetic mouse model. These findings may have significant potential to advance stem-cell-based therapeutics.
From human amniotic fluid.
Council preliminary evaluations in 2005 : Not specifically evaluated.
Update : De Coppi, P., et al., Isolation of amniotic stem cell lines with potential for therapy, Nature Biotechnology, 25: 100-106 (2007)
Abstract : Stem cells capable of differentiating to multiple lineages may be valuable for therapy. We report the isolation of human and rodent amniotic fluid-derived stem (AFS) cells that express embryonic and adult stem cell markers. Undifferentiated AFS cells expand extensively without feeders, double in 36 h and are not tumorigenic. Lines maintained for over 250 population doublings retained long telomeres and a normal karyotype. AFS cells are broadly multipotent. Clonal human lines verified by retroviral marking were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and hepatic lineages. Examples of differentiated cells derived from human AFS cells and displaying specialized functions include neuronal lineage cells secreting the neurotransmitter L-glutamate or expressing G-protein-gated inwardly rectifying potassium channels, hepatic lineage cells producing urea, and osteogenic lineage cells forming tissue-engineered bone.
These last two papers show that multipotent, if not pluripotent, human stem cells may exist in a readily available, ethically unproblematic form without the need to do any exotic genetic engineering or cell biology.