Stem Cells Cloning and Therapeutic Potential.

Stem cells have huge potential to transform current manner in which medicine is practiced. Rather than treating diseased cells with medicines and antibiotics, stem cells can just replace the diseased cells with healthy cells. But it will take time before this research gets translated to the clinic. At present, various types of stem cells like human embryonic stem (hES) cells, induced pluripotent stem (iPS) cells, fetal stem cells, adult tissue-specific stem cells (HSCs, MSCs, etc.), very small embryonic-like stem cells (VSELs) and related technologies like therapeutic cloning are subject to extensive research. Clinicians appear to be in a hurry to apply the stem cells to their patients and there is a huge industry banking stem cells for future autologus use. However, the scientifc community is still not sure which is the best stem cell candidate for regenerative medicine. The chapter provides an update on various fronts and also discusses whether there exists a need to bank stem cells for future use. The author is puzzled by realizing as to what needs to be repaired/ regenerated-the stem cells or their microenvironment ‘niche’!

Nuclear reprogramming and epigenetic rejuvenation.

William Harvey’s motto Ex ovo omnia (‘All from the egg’) on the frontispiece of his treatise On the generation of living creatures (1651) was well chosen and extraordinarily prescient. Centuries later, the egg was shown to have a striking capacity for bringing forth life – life produced by experimental manipulation, ‘animal cloning’, outside the normal physiology of fertilization (Gurdon and Byrne 2005). Cloning through somatic cell nuclear transfer (SCNT) showed that the restriction of developmental potential during cellular differentiation is the result of epigenetic changes in gene expression rather than through loss of DNA – although certain lineages, such as B- and T-cells, are known to undergo programmed DNA rearrangements (Hochedlinger and Jaenisch 2002). It is the reversal of these epigenetic changes during ‘reprogramming’ of the specialized adult nucleus within the reconstructed embryo that results in its re-acquisition of developmental potential and the consequent recapitulation of development, ultimately giving rise to a cloned newborn. A commonly held defi nition is that nuclear reprogramming by SCNT is the process by which a specialized nucleus reacquires developmental potential (Singh 1999). However, nuclear reprogramming is much more than this. It is a manylayered process. Intimately associated with developmental reprogramming of the specialized adult nucleus to an earlier, embryonic, totipotent state is age reprogramming; the ageing ‘clock’ of the transferred nucleus is reset back to zero; an old cell can give rise to newborn clone (Wilmut et al. 1997). This begs the question of whether age reprogramming can be separated from developmental reprogramming. Being able to reprogramme the ageing clock in isolation, while maintaining the differentiated state of a cell, would essentially mean that the cell is made young again: rejuvenated. Clearly, should this be achieved, the consequences would be profound (fi gure 1). Nuclear reprogramming observed in ‘classical’ animal cloning (where adult cells are reprogrammed to an embryonic state after SCNT) has recently been recapitulated in vitro by the generation of embryonic-like induced pluripotent stem cells (iPS cells; Takashi and Yamanaka 2006). Induction of iPS cells allows the process of epigenetic rejuvenation of adult cells to embryonic cells, as seen after SCNT, to be studied in a well-defi ned system (Surani and McClaren 2006). However, certain features of the rejuvenation seen in classical cloning are likely to differ from that seen in iPS cell generation. For one, telomeres, whose shortening is seen as a key characteristic of ageing cells, are ‘rejuvenated’ by telomerase during iPS cell induction from old somatic cells (Marion et al. 2009). This mechanism is unlikely to be the major mechanism for ‘rejuvenating’ telomeres in eggs after SCNT. During the early cleavage divisions, telomeres are lengthened by a telomere sister-chromatid exchange recombination mechanism that is peculiar to this stage of development and is under the sole control of the maternal cytoplasm, as it is unaffected by the absence of a paternal chromosomal complement (Liu et al. 2009). Thus, the mechanism(s) and pathway(s) of rejuvenation that result from SCNT, which have yet to be uncovered, are likely to be different from those operating during the generation of iPS cells. Notwithstanding these differences, SCNT or introduction of ‘reprogramming factors’ into somatic cells both appear to direct developmental reprogramming and age reprogramming seamlessly: age reprogramming does not take place without de-differentiation into embryonic cells (developmental reprogramming). But can these intimately associated aspects of nuclear reprogramming be disentangled? While it seems hardly possible, recent work indicates that age reprogramming might indeed be separable from developmental reprogramming. Differentiation of myelomonocytic progenitors into macrophages involves an exit from the cell cycle.

Microtubule distribution in somatic cell nuclear transfer bovine embryos following control of nuclear remodeling type

This study was conducted to evaluate the microtubule distribution following control of nuclear remodeling by treatment of bovine somatic cell nuclear transfer (SCNT) embryos with caffeine or roscovitine. Bovine somatic cells were fused to enucleated oocytes treated with either 5 mM caffeine or 150 micrometer roscovitine to control the type of nuclear remodeling. The proportion of embryos that underwent premature chromosome condensation (PCC) was increased by caffeine treatment but was reduced by roscovitine treatment (p < 0.05). The microtubule organization was examined by immunostaining beta- and gamma-tubulins at 15 min, 3 h, and 20 h of fusion using laser scanning confocal microscopy. The gamma-tubulin foci inherited from the donor centrosome were observed in most of the SCNT embryos at 15 min of fusion (91.3%) and most of them did not disappear until 3 h after fusion, regardless of treatment (82.9-87.2%). A significantly high proportion of embryos showing an abnormal chromosome or microtubule distribution was observed in the roscovitine-treated group (40.0%, p < 0.05) compared to the caffeine-treated group (22.1%). In conclusion, PCC is a favorable condition for the normal organization of microtubules, and inhibition of PCC can cause abnormal mitotic division of bovine SCNT embryos by causing microtubule dysfunction.