Pluripotent Very Small Embryonic-Like Stem Cells Get Discarded During Cord Blood and Bone Marrow Processing

W e read the comments raised by Ivanovic [1] on our recently published article [2] and thank him for accepting the work as an important technical note. Presence of very small embryonic-like stem cells (VSELs) or VSELs-like cells has been recently reported by several groups in bone marrow (BM), umbilical cord blood (UCB), and also in many other nonhematopoietic tissues [3]. Therefore, we expected an interest in the article and are indeed glad that it caught his attention and motivated him to comment on the work.

A major point of our article was that VSELs are invariably discarded while processing cord blood for banking purpose or while processing BM for autologous stem cell therapy during the volume reduction step. This is an important observation and should be noted by various cord blood banks, stem cell biologists, and policy makers. This observation is important if one wants to employ BM- or UCB-derived cells for regenerative medicine for indications like myocardial infarct, stroke, diabetes and so on. Despite earlier claims, it is now understood that hematopoietic stem cells do not transdifferentiate into other tissues and whatever effect is observed after UCB or BM mononuclear cell therapy is possibly due to paracrine effects or related to the presence of a novel “pluripotent” stem cell population, that is, the VSELs.

Ivanovic also raised an important question about pluripotency of stem cells, and in general, about pluripotency of stem cells isolated from the adult tissues (VSELs). It is a fair question and conceptually, pluripotency of stem cells is defined on the basis of various characteristics listed in the Table 1. We expect that pluripotent stem cells should display high nuclear-cytoplasmic ratio, possess open type of chromatin (euchromatin), express pluripotency markers such as Oct-4 and SSEA, express bivalent domains, and differentiate in vitro into cells from all 3 germ layers. On the other hand, 2 other criteria have been proposed based on work on embryonic pluripotent stem cell lines as their ability to form teratomas and to complete blastocyst development.

As evident, at least murine VSELs express most of these criteria except the ability to form teratoma and complement blastocyst development. One possible reason could be that Mother Nature has evolved mechanisms so that the pluripotent VSELs that normally exist in adult body tissues are extremely quiescent and serve as a backup pool of stem cells to maintain tissue homeostasis and at the same time we should be protected from risk of teratoma formation. To support this, it has been shown that murine VSELs, like primordial germ cells epigenetically modify expression of developmentally important imprinted genes such as Igf2-H19, Rasgrf1, Igf2R, and Kcnq1 [4], which renders them quiescent in adult tissues and protects from uncontrolled proliferation. Whether this is also a case for human VSELs requires further studies.

VSELs were initially isolated from murine BM and have been characterized to be positive for crucial pluripotency marker Oct-4, besides several other markers. The expression of Oct-4 in VSELs has been reported by several groups and confirmed by demonstrating transcriptionally active chromatin in Oct-4 promoter [4]. VSELs exist in various mouse and human organs [3], have been well characterized, and are capable of differentiating into all 3 lineages, supporting their true pluripotent character. Murine VSELs form embryoid bodies like embryonic stem (ES) cells when cultured over C2C12 supportive cell line, can differentiate into hematopoietic stem cells (HSCs) after coculture over OP-9 stroma cells and reconstitute murine BM after total body irradiation. They have also been shown to differentiate into neural cells and cardiomyocytes in rodents. Earlier, McGuckin et al. [5] have isolated VSELs (2–3 μm in diameter) from UCB, which are CD45−, CD33−, CD7−, CD235a−, and CD133+ and express pluripotent markers Oct-4 and Sox-2. They further successfully differentiated VSELs into hepatocytes and neural progenitors by sequential introduction of growth factors/cytokines under defined serum-free culture conditions. We detected VSELs also in the umbilical cord tissue (Wharton's jelly) as a subpopulation among the mesenchymal stem cells (MSCs). VSELs are mobilized into peripheral blood in response to injury/stress [3] including acute myocardial ischemia and infarct, stroke, and burns in animal models and in humans, thus suggesting a role in regeneration and homeostasis.

Work on VSELs and, in particular, on human VSELs is yet in its infancy and more experimental evidence in relation to their functionality and regenerative potential will emerge in due course. We believe that similar to that in mouse, VSELs also exist in various body tissues in humans, and their differentiated fate is guided by the somatic microenvironment [6]. We have characterized BM/UCB VSELs as CD45−/Lin−/CD34+/Oct4+/Nanog+/Rex+/Tert+/SSEA4+/CXCR4+/SSEA1+/Fragilis+, which suggests their nonhematopoietic, pluripotent, and migratory properties along with a common origin from precursors of primordial germ cells, as has been suggested earlier. Rather than criticizing each other, we should work together to exploit the potential of VSELs. Indeed, we propose that like A_darkspermatogonial stem cells in testis and ovarian germ stem cells (OGSCs) in ovary (with cytoplasmic OCT-4), HSCs and MSCs (with cytoplasmic OCT-4) in UCB, BM, and umbilical cord tissue are descendants “progenitors” derived from the VSELs (with nuclear OCT-4). HSCs and MSCs have been successful during BM transplantation because they are “committed” progenitors to make blood cells to reconstitute the BM, but they possibly may not have the ability to transdifferentiate into other lineages as compared with VSELs, which being pluripotent are highly “plastic” in nature. Method to isolate VSELs from BM and cord blood reported by us is simple [2], whereas, isolation of VSELs by sorting requires expertise and anyone who wishes to work on these cells must strictly follow already established protocols [3]. We will like to conclude by posing 3 questions (i) do we truly know what types of non-hematopoietic stem cells are present in UCB and BM, (ii) which stem cells are effective during BM/UCB stem cell clinical trials in regenerative medicine? (iii) what is the need to reprogram somatic cells to make induced pluripotent stem cells (iPS) cells when pluripotent stem cells (VSELs) exist in various adult body tissues? It is indeed time for the stem cell community to retrospect and reflect!