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

76 Blood Cell – An Overview of Studies in Hematology

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].

with HSCs first fetal liver and subsequently BM [37].

migratory germ layer.'' [37]

*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

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

*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 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.** These VSELs exhibited various pluripotent markers, like CD45- , CD133+ SSEA-4+. They also 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 mesentry to the gonadal ridges to become a source of germ cells.


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

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

minimal improvement. This may be explained since fate restricted progenitors HSC and MSC may have limited trans-differentiation ability. The pluripotent VSELs have maximum 'plasticity' and regenerative potential but are getting discarded unknowingly. This raises a valid question on the success of BM transplantation to treat blood related diseases. This success could be accounted for by the differentiation ability of progenitor cells into blood cells.

VSELs in Bone Marrow and Cord Blood 79

microenvironment. Recently it has also been reported that VSELs resist radiotherapy (because of their quiescent nature) that destroys all actively dividing stem cell population in the bone marrow [43]. The somatic microenvironment is also compromised by the radiotherapy. Thus

Existence of two stem cell populations in various adult body tissues is an interesting concept put forth by Li and Clevers [58]. They proposed that both quiescent (out of cell cycle and in a lower metabolic state) and active (in cell cycle and not able to retain DNA labels) stem cell subpopulations may coexist in several tissues like gut epithelium, hair follicle, bone marrow etc. We have generated data to show that similar two distinct populations of stem cells exist in mammalian gonads also. Interestingly similar stem cell biology persists in the mammalian gonads irrespective of sex and is possibly an evolutionarily conserved phenomenon as we

We have reported for the first time the presence of a distinct population of VSELs with nuclear OCT-4 in adult mouse [48] and human [47] testis, located towards the basement membrane of the seminiferous tubules. Besides, we also detected a progenitor stem cell population with cytoplasmic OCT-4, which was slightly bigger and had abundant cytoplasm. These cells showed extensive proliferation with cytoplasmic bridges as cords. As these cells differentiated further, the cytoplasmic OCT-4 was gradually lost. Interestingly the VSELs were found resistant to busulphan treatment which otherwise destroyed the dividing progenitors, haploid cells and damaged the somatic niche. Thus, it is evident that like the earlier report on bone marrow VSELs, gonadal VSELs are also resistant to oncotherapy. VSELs possibly undergo asymmetric cell division to give rise to progenitors, which undergo

A gentle scraping of the adult ovary surface (mouse, rabbit, sheep, monkey and human) with a sterile blade releases stem cells in a Petri dish [50]. On H & E staining, two distinct stem cell populations can be easily detected based on their size and differential OCT-4 staining pattern. The smaller stem cell population are smaller than the RBCs and exhibit nuclear OCT-4 whereas the slightly bigger population exhibits cytoplasmic OCT-4. Like cords in the testis, in the ovary we observed the presence of germ cell nests with cytoplasmic continuity representing extensive proliferation of progenitor stem cells. These stem cells were present in peri- menopausal human ovary and also persisted in mouse ovary after busulphan treatment. Like in the testis, the functionality of ovarian stem cells is also affected

Three weeks culture of peri-menopausal ovarian stem cells produces oocyte-like structures, embryo-like structures *in vitro* [50]. Thus the stem cells retain their functionality but are

although the VSELs persist, they are unable to reconstitute the bone marrow.

have reported the same in mice, rabbits, sheep, monkey and humans [48, 50].

clonal expansion and may further differentiate into sperm (Figure:5)

unable to differentiate because of a non-supportive niche.

**6. VSELs in mammalian testis** 

**7. VSELs in mammalian ovary** 

by a compromised niche.

While doing immunolocalization studies to detect OCT-4 positive cells, we found the VSELs express nuclear OCT-4 whereas a slightly bigger cell in the 'buffy coat' collected from both the cord blood and bone marrow exhibited cytoplasmic OCT-4 (Figure:4). These are possibly the most immediate progenitors 'descendants' from VSELs. We also conducted immunolocalization studies on umbilical cord tissue in the region of Wharton's jelly which is rich in MSCs. Results show that the MSCs had cytoplasmic OCT-4 like HSCs and that there was a distinct subpopulation of small cells with nuclear OCT-4 and were the VSELs, based on their size (Figure:4). On a similar note, when we did immunolocalization of mouse bone marrow stem cells, we observed that the MSCs with typical fibroblast like morphology have cytoplasmic OCT-4 along with VSELs with nuclear OCT-4. The MSCs showed a very heterogeneous staining pattern. Only a sub population of MSCs were positive whereas other MSCs totally lacked cytoplasmic OCT-4. This possibly shows different differentiation state since as the cell gets more committed, cytoplasmic OCT-4 is no longer required.

**Figure 4.** Immunolocalization of Oct-4 in umbillical cord tissue

Thus we concluded that the bone marrow compartment comprises of pluripotent VSELs and their immediate descendants like HSCs and MSCs. Also that the most primitive stem cell in the bone marrow is a pluripotent VSEL as shown in Figure 2. Being the most primitive stem cell in the BM, we hypothesize that VSEL will show best engraftment post transplantation and also will be best vehicle for gene therapy.

VSELs possibly undergo asymmetric cell division to self- renew and give rise to progenitors which further expand and differentiate to committed cell types. VSELs remain relatively quiescent throughout life, maintain long telomeres and are possibly the normal body stem cells which give rise to cancer stem cells (CSC) under certain unfavourable conditions. We propose that this transformation of a VSEL into CSC occurs due unidentified changes in the microenvironment. Recently it has also been reported that VSELs resist radiotherapy (because of their quiescent nature) that destroys all actively dividing stem cell population in the bone marrow [43]. The somatic microenvironment is also compromised by the radiotherapy. Thus although the VSELs persist, they are unable to reconstitute the bone marrow.

Existence of two stem cell populations in various adult body tissues is an interesting concept put forth by Li and Clevers [58]. They proposed that both quiescent (out of cell cycle and in a lower metabolic state) and active (in cell cycle and not able to retain DNA labels) stem cell subpopulations may coexist in several tissues like gut epithelium, hair follicle, bone marrow etc. We have generated data to show that similar two distinct populations of stem cells exist in mammalian gonads also. Interestingly similar stem cell biology persists in the mammalian gonads irrespective of sex and is possibly an evolutionarily conserved phenomenon as we have reported the same in mice, rabbits, sheep, monkey and humans [48, 50].
