Human embryonic stem cells Preclinical perspectives
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Human Embryonic Stem Cells: Preclinical perspectives Surjya Narayan Dash, Kanchan Sarda, Kaushik Deb Embryonic Stem Cell Program, Manipal Institute of Regenerative Medicine, #10 Service Road, Domlur, Bangalore 560071, India Email: firstname.lastname@example.org email@example.com Deb et al., 2008 (Journal of Translational medicine)
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Human embryonic stem cells (hESCs) Cell replacement therapies (CRTs) Inner cell mass (ICM) in vitro fertilization (IVF) Somatic cell nuclear transfer (SCNT) Preimplantation genetic diagnosis (PGDs) Blastomere-like stem cells (BLSCs) Embryonic-like stem cells (ELSCs) Fluorescence Activated Cell Sorting (FACS) Assisted reproductive technologies (ART) Human nuclear transfer ESC (hNT-ESCs) Some Important Key words
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Sir Martin Evans has recently been honored with the Nobel Prize for Physiology and Medicine (2007) for his contribution towards development of animal models of disease through ESC mediated gene targeting. Human embryonic stem cells were first derived by Thompson’s group in 1998 and are usually derived from the inner cell mass (ICM) of blastocyst stage embryos that are left over after in vitro fertilization (IVF) and after embryo donations Fig.1 Sir Martin Evans receiving the Nobel prize
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Not much has been achieved in turning them into safe therapeutic agents Human embryonic stem cells (hESCs) have discussed in public and scientific communities for their potential in treating diseases and injuries
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Debate on hESCs therapy Debate about the benefits and drawbacks of adult vs. hESC use in therapies. The use of human embryonic stem cells (hESCs) in cell replacement therapies (CRTs) has been limited The use of human embryonic stem cells (hESCs) in cell replacement therapies (CRTs) has been limited due to several technical and ethical issues
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Barriers to bringing hESCs to clinic Changes in their epigenetic profiles Chromosomal aberrations during their establishment and maintenance Post transplantation challenges like risk of tumors Immune-rejections
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Need for xeno-free culture systems Human ES cells are generally grown In a medium containing animal serum as a source of nutrients and growth factors On mouse-derived fibroblasts as feeder layer Use of any cell based therapeutic agent in human must be free of animal contaminations which may contain certain pathogens or xenogens that can trigger immune reactions after transfer to a host .
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hES cell colony grown in Matrigel hES cell colony grown in Mouse feeder Fig.2
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Expression of a nonhuman sialic acid Neu5Gc and presence of murine viruses are two concerns in existing hESCs grown in presence of animal products or feeders. Replacement of animal serum with human serum has been reported to reduce the expression of Neu5Gc in the hESCs, also Amit et al (2005) have reported the absence of murine leukemia virus in a number of hESC lines maintained on mouse feeders
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Risk of tumors Transplantation of hES cell based therapies involves the risk of tumor formation arising from undifferentiated population of the transplanted cells. Studies with both ESCs and ES derived differentiated cells have shown that they can form teratocarcinomas in adult mice if injected subcutaneously, intramuscularly or into the testis. Presence of even one undifferentiated cell may potentially lead to teratomas, a cancerous tumor which is derived from germ cells and can from all the three germ layers.
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Genetic instability Questions on the suitability of ESCs for transplantation purpose is raised because of the observed genetic instability of cloned cells and extreme inefficiency of the process. Cloned animals like Dolly give the outward appearance of full health, but the probability of their having numerous genetic defects is very high. Hochedlinger and Rudolf Jaenisch (2002) showed that in mice, the reprogramming of the inserted genetic material by the embryonic cells proceeded in a very unregulated way .
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Transplant rejection The immune system tends to reject the transplanted ESCs as 'foreign'. This rejection can be inhibited by the use of immunosuppressive drugs which can have serious side effects. Alternate approaches using homolologous recombination techniques can allow the host immune system to recognize and mark the ESCs as 'self'. Elimination of MHC class I and II gene loci is also proposed, though this would be technically challenging and would be clinically problematic
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Epigenetic reprogramming and culture adaptation Two major causes for epigenetic changes in hESCs have been identified. The epigenetic changes in preimplantation embryos used for derivation of the hES cell lines Epigenetic changes during their maintenance in the culture over time
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Chromosomal abnormalities during prolonged culture Several reports also indicate that these cells acquire chromosomal abnormalities or karyotype aberrations during prolonged culture in parallel with epigenetic changes. Such adaptations may result in enhanced cloning efficiencies after plating single cells . A reduced tendency for apoptosis and is expected to have a reduced capacity for differentiation which is difficult to assess quantitatively. A recent report by Baker et al., (2007) demonstrates accumulation of specific chromosomal aberrations within several well-established hESC lines over time.
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Embryonic stem cell based therapies: advances What may have appeared to be impossible with ESC research several years ago is gradually turning into reality. Efforts are being made to use this technology, to modify the ESCs for use in delivery of genes and other factors to dying motor neurons. Generation of patient specific human nuclear transfer ESC (hNT-ESCs) lines is a strategy that may circumvent the problem of immune-rejection which is the greatest challenge in CRTs. The implications of transferring mitochondrial hetroplasmic cells, which might contain aberrant epigenetic gene expression profiles, are also of concern. Allogenic mitochondria present in the NT-ESC derived cells could be recognized by the host immune system, leading to disrupted mitochondrial membrane potential that induces apoptosis
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The mitochondrial genome is also known to encode a number of transplantation antigens that could trigger a immune response for the host tissue following engraftment. Pathenogenetically activated embryos has been proposed for the creation of female haploid ESC lines. These cells could serve as an autologous source of cells for producing differentiated cell types to treat women suffering from diseases like Type 1 diabetes or spinal cord injuries. Revazova et al., (2007) has reported the development of six patient specific stem cell lines from parthenogenetic blastocysts which is a better prospective for clinical trials .
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Trivedi et al., (2006) has reported a unique technique for tolerance induction using nuclear transfer (NT)-hESC-induced hematopoietic chimerism with synergistic use of adult bone marrow . Although these reports are very promising a great deal of preclinical research still needs to be undertaken before the envisioned therapeutic potential of ESCs can be translated to the bedside.
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Derivation of hESCs in “Embryo-friendly” ways Reprogramming of adult cell nucleus (iPSCs) ESCs from embryo like entities ESC lines from single blastomeres ESC lines from induced somatic cell dedifferentiation Embryonic like stem cells from alternative sources Alternates to blastocyst derived hESCs:
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Table : A list of animal injury and disease models where hESCs have been shown to be effective
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hES cells derived in a embryo friendly ways Pure population of differentiated cells With out chromosomal abnormality Xeno-free culture of hESCs Possibilities of clinical trials Clinical prospective of hES cells Fig.3 Human embryo at blastocyst stage ES cell colonies derived from inner cell mass
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