Mahendra Rao, Vice President for Regenerative Medicine at the New York Stem Cell Foundation, USA, and Editorial Board member for Stem Cell Research & Therapy, looks back at two decades of progress in pluripotent stem cell research, highlighting some of the key advances that may inform future progress in stem cell therapy*.


Pluripotent stem cell (PSC) based therapy is a very young field. Although we have known how to culture mouse embryonic stem cells for many years we were unable to adapt those techniques to growing  human cells for decades. Pioneering work performed in the lab of James Thomson that built on work done by groups led by Ariff Bongso, Joseph Itskovitz-Eldor and others enabled investigators to develop techniques that allowed us to make non-human primate embryonic stem cells (ESCs) and subsequently ESC lines from human blastocysts (Science, 1998, 282:1145–1147). Early relatively inefficient techniques were rapidly improved on such that making ESC lines is relatively straightforward and more than a thousand lines have been derived worldwide with at least 200 lines being registered in the US National Institutes of Health (NIH) registry, which evaluates whether the lines are eligible for Federal funding.

Since the initial derivation in 1996, several technical advances have enabled widespread use of ESC based technology. This includes techniques for deriving lines without destroying the embryo, deriving lines from different stages of pluripotency  and deriving parthenogenetic lines (Stem Cells Dev, 2008, Feb, 17(1):1-10). These advances coupled with advances in techniques in culturing cells and identifying the growth factors that are required for maintaining undifferentiated cells suggested that one could derive lines that fulfilled the US Food and Drug Administration (FDA) regulations for being clinically compliant and could be used as starting material for  generating differentiated cell products (Gene Therapy, 2008, 15, 82–88 and Nat Biotechnol, 2009, 27, 606-613). Indeed several companies have developed such clinically compliant lines and offered them to researchers for the development of their individual end products. Companies like Geron and ACT have shown that end products can be manufactured without risk of containing enough undifferentiated cells to form teratomas.

Although cells derived from ESCs could be considered safe there still remained the issue of immune matching. What had become clear from the work done in the cell and organ transplant fields was that there were no truly immune privileged sites and that most cells would provoke rejection unless immune suppression techniques were used. Although some investigators have argued that life time immune suppression will be required  the emerging consensus seemed that at the very least short term suppression would be necessary. Even such limited immunosupression however, introduces its own risks and difficulties in designing trials and performing adequate follow-up studies. Investigators attempted to resolve this issue in several different ways. One strategy was to consider building banks of human leukocyte antigen (HLA) matched cells. Estimates as to the size of such banks was debated but it appeared possible though expensive (Stem Cells and Development, 2012, 21, 13: 2364-2373 and Cell Stem Cell, 2013, Oct, 13(4):382-4).

Other investigators suggested engineering techniques where the entire HLA-locus was reorganised to obtain universal donor lines while yet other groups suggested nuclear transfer techniques that had been pioneered in frog embryos by John Gurdon and his colleagues. Early success in a number of species led to cloning of ‘Dolly the sheep’, ‘SNUPI the dog’ and to cloning pigs, however there were difficulties with extending these techniques to humans. There were issues with the technique and maintaining the integrity of the mitotic spindle that would allow successful cell division and growth. The recent work of groups led by Dieter Egli and Maisam Mitalipova have shown that these hurdles can be overcome (Cold Spring Harb Perspect Biol, 2011 Jun 1;3(6); Nature, October 2011, 478: 70–75; Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer, June 2013, Vol 153, 6:1228–1238). However, although there have been steady improvements in these techniques it is perhaps fair to say that the process is still laborious, time consuming and relatively inefficient. Overall although there has been tremendous progress in a short span of fifteen years these advances have also highlighted the practical difficulties of using ESC derived products for therapy. This coupled with the residual ethical concerns that some groups have expressed led to a focus on using existing lines for non therapeutic uses, understanding basic biology of stem cells and the pluripotent state and exploring other sources of pluripotent cells.


Paths to pluripotency: In some species PSCs may make trophoblasts. PSCs have been derived from blastocysts at various stages of development, from individual blastomeres, and from donors with hereditary defects. Using unfertilised eggs researchers have been able to derive PSC lines by somatic cell nuclear transfer (SCNT). Researchers have shown that germ lineages can be cultured to induce pluripotency, whilst others argue that stem cells persist in the adult and may be harvested from all tissues. VSEL (very small embryonic stem cell like cells) have gained prominence recently. Perhaps the most robust and technically straightforward way to make PSCs has been the induced PSC (iPSC) method. PSCs from flash frozen tissue, urine and even postmitotic cells have also been reported. ESC (embryonic stem cells), PGD (preimplantation genetic diagnosis), EBD (embryoid body dervied), TSC (trophoblast stem cells), PGC (primordial germ cells), PPSC (pluripotent stem cells). Image source: Mahendra Rao, New York Stem Cell Foundation, USA.


Cells derived from germ cells, embryoid body derived (EBD) cells, were described and work suggested that the gamete lineage may retain pluripotency (Nature Biotechnology, 2006, 24, 663-664). Oogonial, testicular and spermatogonial stem cells  have also been described  (see for example Nature, April 2006, 440, 1199-1203). In addition to these two populations, other investigators have suggested that stem cells (non-ESC), though tissue specific in their contribution during development, can be induced to reacquire additional capabilities by manipulating cell culture conditions or exposing them to drugs that alter nuclear structure or exposing them to cytoplasm from ES cells, unfertilised eggs, or germ cell tumours. These transdifferentiated or dedifferentiated cells, while differing from ES cells, may be functionally equivalent for therapeutic applications. The possible existence of such cells has fanned an ethical debate on determining whether deriving new ES lines is more appropriate or whether one should focus on dedifferentiating or transdifferentiating adult stem cells. Some of the excitement was dampened  when the initial positive results  were not replicated or alternative explanations such as cell fusion  appeared to explain the results adequately.

Other groups argued that every tissue contained pluripotent stem cells that were like ESCs but did not form tumours. The best described of such cells is the VSEL or very small adult derived embryonic stem cell like cell. VSEL cells gained some notoriety when they were recommended by the church and because they appeared to offer the best of all worlds – they was no ethical issue, they were autologous and thereby resolved the immune issues of transplantation, they were non-tumourigenic and did not form teratomas, and they were present in sufficient numbers that they could be readily harvested by  simple modification of standard procedures (Journal of Autoimmunity, May 2008, Vol 30, 3:151–162; Wound Repair Regen, 3:457–468; Stem Cells, 2009, 27:3053–3062). In more recent years there have been skeptics as many laboratories could not replicate the results, the benefits did not seem as spectacular as first described, and propagating and differentiating these cells in culture seemed difficult.

The field of pluripotent stem cell biology underwent a seismic change when Shinya Yamanaka along with his colleague Kazutoshi Takahashi announced that they could indeed generate pluripotent cells from adult tissue (Cell, 2006, 126:663-67). It required more than simple manipulation of culture conditions but they were able to show that a robust reliable process of using four genes alone was enough to direct the differentiation of adult cells to an ESC like state. The process of induced pluripotent stem cell (iPSC) generation was rapidly replicated by hundreds of laboratories all over the world and the results were compelling. Most laboratories could generate iPSCd, the cells behaved like ESCs and such cells could be obtained from multiple species, multiple starting cell populations and at different stages in development. It was very clear that Yamanaka and colleagues had tapped into a fundamental biological process and shown how it  could be done in a relatively straightforward way. The importance of this discovery was honored by the award of a Nobel Prize to Yamanaka jointly with Gurdon, who had first shown that reprogramming should be possible.

As this overview perhaps shows progress has been rapid and in less that two decades we have come from learning how to make human ESCs to discussing how to produce ESC like cells from every individual in a cost effective way. A few brave pioneers have already manufactured cells from pluripotent stem cells and patients have been treated with such cells in early phase I trials. Plans are underway to initiate similar studies with iPSCs and several investigator initiated trials are under development. Overall the PSC field has been shaped not just by technology and scientific breakthroughs but also by the enormous expectations that people have for this novel type of therapy.


*The opinions expressed in this article represent the views of Mahendra Rao and do not represent the policy of the organisation(s) with whom he is affiliated.

More about the researcher(s)

  • Mahendra Rao, Vice President for Regenerative Medicine,  New York Stem Cell Foundation, USA.

    Mahendra Rao

    Mahendra Rao is Vice President for Regenerative Medicine at the New York Stem Cell Foundation, USA. He received his medical degree from Bombay University, India, and his PhD in developmental neurobiology from the California Institute of Technology, USA. Rao pursued his postdoctoral training at Case Western Reserve, USA and then established a career as an… Read more »

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