**6. VSELs in mammalian testis**

78 Blood Cell – An Overview of Studies in Hematology

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.

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.

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

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

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

and also will be best vehicle for gene therapy.

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

## **7. VSELs in mammalian ovary**

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 by a compromised niche.

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 unable to differentiate because of a non-supportive niche.

VSELs in Bone Marrow and Cord Blood 81

Several years of cancer research suggests that cancers begin with genetic changes that occur over a period of 15 to 20 years and in few cases a link to chronic inflammation has been proposed e.g. in case of ovarian cancers, Barrett's esophagus etc. However, emerging literature suggests that quiescent VSELs distributed in various organs may be a cellular origin of cancer development. In 1855 Virchow proposed the embryonal rest hypothesis of tumor formation, based on histological similarities between tumors and embryonic tissues. This theory was later expanded by other pathologist including Julius Conheim, who suggested that tumors develop

Recently identified VSELs in various adult body tissues display morphology and markers characteristics as the pluripotent embryonic stem cells. These cells could support Virchow's concept of an embryonic origin of cancer. Possibly the somatic niche, which keeps the VSELs in a quiescent stage under normal circumstances, undergoes some changes which push the

Wang et al [60] recently reported that persisting embryonic cells in adult mice and humans at the squamo-columnar junction are possibly the source of Barrett's metaplasia and that it does not arise from mutant cells. They proposed that certain precancerous lesions, such as Barrett's, initiate not from genetic alterations but from competitive interactions between cell lineages driven by opportunity. Similarly, almost 90% of ovarian cancers arise from the ovary surface epithelium which is also the niche for ovarian stem cells. It is being proposed that ovarian niche gets compromised with age leading to menopause [61, 62] and also to cancer. It is essential to dissect out age related changes which lead to menopause and how they differ from those which lead to cancer. OCT-4, characteristic marker of VSELs is also a very good

marker with high sensitivity and specificity for testicular germ cell tumors as well [63].

Cancer stem cells and VSELs with embryonic characteristics have a lot of similarities in terms of markers, telomere length, and resistance to radiotherapy; thus it may be proposed that VSELs transform into CSCs when certain not so well understood changes occur in the microenvironment. It is possible that inflammation may alter the niche where the VSELs reside. It is highly unlikely that a somatic cell which is relatively senescent and has short telomeres will dedifferentiate and acquire long telomeres to transform into a cancer stem cell. Keeping this in mind, because of a defect in stem cell in the bone marrow due to an altered niche – defective stem cell divisions occur and differentiation of such altered cells results in appearance of chromosomal defects in mononuclear cells picked by standard

Identification of VSELs in adult tissues also opens new areas of investigation to elucidate how these cells contribute to the development of poorly differentiated tumors. Studying the biology of normal stem cells may help us to better understand the biology of cancer, and explain its resistance to radio-chemotherapy, ability to an unlimited proliferation and

from residual embryonic remnants lost during developmental organogenesis [59]

quiescent VSELs to an actively dividing state i.e. the tumor.

**9. VSELs and cancer** 

cytogenetic studies.

establishment of distant metastases.

**Figure 5.** Revised scheme for premeiotic development of germ cells in adult human testis
