**4. Very small embryonic like stem cells (VSELs)**

VSELs were identified by Ratajczak and group in 2006 by multi parameter sorting in adult murine BM. They express several morphological (e.g., relatively large nuclei containing euchromatin) and molecular (e.g. expression of SSEA-1, Oct4, Nanog, Rex1) markers

characteristic for embryonic stem cells (ESCs) [33]. The morphology of the cells was investigated using transmission electron microscopy which showed their distinctive morphology and size differentiating VSELs from HSC in particular in terms of size (3–6 μm vs. 6–8 μm for HSC), chromatin structure and nucleus/cytoplasm ratio. Based on their small size, presence of PSC markers, distinct morphology (open-type chromatin, large nucleus, narrow rim of cytoplasm with multiple mitochondria) and ability to differentiate into all three germ layers, including mesoderm-derived cardiomyocytes, these cells were named very small embryoniclike stem cells. The true expression of Oct-4 and Nanog in BM-derived VSELs (BM-VSELs) was recently confirmed by demonstrating transcriptionally active chromatin structures of Oct4 and Nanog promoters. A mechanism based on parent-of-origin-specific reprogramming of genomic imprinting that keeps VSELs quiescent in a dormant state in tissues has been described. VSELs highly express Gbx2, Fgf5, and Nodal, but express less Rex1/Zfp42 transcript as compared to ESC-D3s what suggests that VSELs are more differentiated than ICM-derived ESCs and share several markers with more differentiated EpiSCs. VSELs also highly express Dppa2, Dppa4, and Mvh, which characterize late migratory PGCs. The expression of germ line markers (Oct4 and SSEA-1) and modulation of somatic imprints suggest a potential developmental similarity between VSELs and germ line-derived primordial germ cells (PGCs) [39, 40].

VSELs in Bone Marrow and Cord Blood 77

, CD133+ SSEA-4+. They also

have also reported that VSELs are mobilized into peripheral blood in response to injury/ stress in animal models [27,54-56] as well as in humans [28-30,57] – thus suggesting a role in

We studied the VSELs in UCB and discarded fraction of BM [46]. Usually the 'buffy coat' obtained after Ficoll-Hypaque centrifugation is considered to be rich in stem cells and used for various studies over several decades. **However, we reported that VSELs settle along with the RBCs rather than getting enriched in the 'buffy coat (Figure:3). Similarly we found that the 'discarded' RBC pellet obtained during initial processing of bone marrow was also rich in VSELs. These results were explained on the basis of buoyancy. The adult stem cells have abundant cytoplasm, are relatively larger and thus observed in the buffy layer whereas the VSELs are the pluripotent stem cells, with high nucleo-cytoplasmic ratio, minimal cytoplasm and thus sink to the bottom of the tube along with the RBCs.**

exhibit other primordial germ cell markers like Stella and Fragillis, thus supporting their origin from the epiblast stage embryo at the same time when PGCs migrate via the dorsal

**Figure 3. Isolation and characterization of VSELs from Cord Blood: A-Separation** of cord blood into

These studies have several implications e.g. the stem cell biologists should ask themselves what is getting banked in the cord blood banks. VSELs unknowingly get discarded and only adult stem cells (and progenitors) including HSCs and MSCs get banked. Similarly autologus stem cell therapy for various indications other than blood related diseases have resulted in

four layers on Ficoll-Hypaque; B-Description of cells observed in each layer separated; C-Immunolocalization studies on MNC (A) and VSEL (B) using polyclonal Oct-4 (40X); D-Markers

characterized on VSELs using Quantitative PCR and immunofluorescence

**5. Our studies on VSELs in cord blood and bone marrow** 

These VSELs exhibited various pluripotent markers, like CD45-

mesentry to the gonadal ridges to become a source of germ cells.

regeneration and homeostasis.

*Developmental Origin of VSELs*: VSELs are epiblast-derived PSCs deposited early during embryonic development in developing organs as a potential reserve pool of precursors for TCSCs and thus this population has an important role in tissue rejuvenation and regeneration. VSELs originate from or are closely related to a population of proximal epiblast migratory Stem Cells (EpiSCs) that approximately at embryonic day (E)7.25 in mice, become specified to PGCs, and egress from the epiblast into extra-embryonic tissues (extraembryonic mesoderm) [41]. VSELs follow developmental route of HSCs colonizing together with HSCs first fetal liver and subsequently BM [37].

Thus PGCs, HSCs, and VSELs form all together a unique highly migratory population of interrelated Stem Cells (SCs) that could be envisioned to be a kind of ''fourth highly migratory germ layer.'' [37]

*Self-renewal and in vitro differentiation of VSELs*: VSELs exist in various mouse organs [42], have been well-characterized and are capable of differentiating into all three lineages, supporting their true pluripotent character. Murine VSELs form embryoid body-like structures in co-cultures over C2C12 supportive cell line [24] and could become specified into HSCs after co-culture over OP-9 stroma cells. VSELs-derived HSCs harvested from these co-cultures reconstitute murine bone marrow after total body irradiation [43]. The Umbilical Cord Blood (UCB)-purified VSELs have also been reported to differentiate into neural cells [44] and after co-culture over OP-9 stroma cells were specified into HSCs similar to murine BM-derived VSELs [45]. Apart from umbilical cord blood and bone marrow, VSELs have also been reported in Wharton's jelly and gonadal tissue [46- 51]. Their presence amongst the MSCs in the Wharton's jelly is in agreement with observations made by other groups that MSCs contain a sub-population of more primitive stem cells [52] or even as postulated by Taichman and group [53] that VSELs are precursors of MSCs. Various studies have also reported that VSELs are mobilized into peripheral blood in response to injury/ stress in animal models [27,54-56] as well as in humans [28-30,57] – thus suggesting a role in regeneration and homeostasis.
