Additional Evidence to Establish Existence of Two Stem Cell Populations Including VSELs and SSCs in Adult Mouse Testes

Kaushik, Ankita; Bhartiya, Deepa

doi:10.1007/s12015-020-09993-6

Cited by 27 publications

(17 citation statements)

References 35 publications

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“…Somatic cells pellet down at this speed, and care is taken not to spin at higher speed; otherwise, they may get damaged and cytoplasm may burst leading to cell death. However, stem cells require higher speed of 1000g to pellet down [8][9][10][11][12][13] (Fig. 1).…”

Section: Main Textmentioning

confidence: 99%

“…b and c have been reproduced from our original article [9] with permission from Springer Nature later centrifugation of the supernatant at 1000g allows enrichment of stem cells. Using this simple and robust approach that can be easily replicated by any lab, VSELs and slightly bigger "progenitors" can be enriched in multiple adult tissues [8][9][10][11][12][13]. Appreciating and incorporating this crucial detail during processing to study stem cells will ensure their detection by single-cell analysis and will empower adult stem cell biology in normal and cancerous tissues and regenerative medicine to progress convincingly.…”

Section: Main Textmentioning

confidence: 99%

“…Ovarian cell isolation at 300g for single-cell analysis [1] Stem cells can be isolated by scraping ovary cell surface [12] Prostate Prostate cell isolation at 200g for single-cell analysis [2] Cardiac Cardiac cell isolation at 100g for single-cell analysis [3] Pancreas Isolation of pancreatic cells and processing for multicolor flow cytometry at 1200 rpm [14] Single cell suspension spun at 1000 rpm and then supernatant at 1000g helped collect VSELs and pancreas stem cells (PSCs) from both pancreas and the islets [9] Testes Single cell suspension spun at 1000 rpm and then supernatant at 1000g to collect VSELs and spermatogonial stem cells (SSCs) [11] Uterus Single cell suspension spun at 1000 rpm and then supernatant at 1000g to collect VSELs and endometrial stem cells (EnSCs) [10] Pancreas, spleen, lungs, gut epithelium, bone marrow, muscles…”

Section: Ovarymentioning

confidence: 99%

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Adult tissue-resident stem cells—fact or fiction?

Bhartiya

2021

Stem Cell Res Ther

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Life-long tissue homeostasis of adult tissues is supposedly maintained by the resident stem cells. These stem cells are quiescent in nature and rarely divide to self-renew and give rise to tissue-specific “progenitors” (lineage-restricted and tissue-committed) which divide rapidly and differentiate into tissue-specific cell types. However, it has proved difficult to isolate these quiescent stem cells as a physical entity. Recent single-cell RNAseq studies on several adult tissues including ovary, prostate, and cardiac tissues have not been able to detect stem cells. Thus, it has been postulated that adult cells dedifferentiate to stem-like state to ensure regeneration and can be defined as cells capable to replace lost cells through mitosis. This idea challenges basic paradigm of development biology regarding plasticity that a cell enters point of no return once it initiates differentiation. The underlying reason for this dilemma is that we are putting stem cells and somatic cells together while processing for various studies. Stem cells and adult mature cell types are distinct entities; stem cells are quiescent, small in size, and with minimal organelles whereas the mature cells are metabolically active and have multiple organelles lying in abundant cytoplasm. As a result, they do not pellet down together when centrifuged at 100–350g. At this speed, mature cells get collected but stem cells remain buoyant and can be pelleted by centrifuging at 1000g. Thus, inability to detect stem cells in recently published single-cell RNAseq studies is because the stem cells were unknowingly discarded while processing and were never subjected to RNAseq. This needs to be kept in mind before proposing to redefine adult stem cells.

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Section: Main Textmentioning

confidence: 99%

Section: Main Textmentioning

confidence: 99%

Section: Ovarymentioning

confidence: 99%

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Adult tissue-resident stem cells—fact or fiction?

Bhartiya

2021

Stem Cell Res Ther

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“…We have recently reported a simple and robust protocol to enrich both the populations of stem cells from mice testes along with their detailed characterization [ 55 ]. FSHR expression was studied in normal testes and after 30 days of busulphan treatment (25 mg/Kg) with and without FSH treatment (10 IU per day for 2 days) and PMSG (10 IU per day for 2 days) [ 52 ].…”

Section: Main Textmentioning

confidence: 99%

“…FSHR expression was studied on adult mouse testicular stem cells by immuno-fluorescence using two different commercial antibodies (Abcam, Santacruz), by Western blot, in situ hybridization using oligoprobes specific for Fshr1 and Fshr3 and by qRT-PCR. Fshr-1 and Fshr-3 expression was also studied in mice testes neonatally exposed to endocrine disruption with estradiol (20 μg/pup/ day on days 5–7) and diethylstilbestrol (2 μg/pup/day on days 1–5) [ 55 ]. Twenty-four hours of treatment to both intact (PMSG 10 IU, Fig.…”

Section: Main Textmentioning

confidence: 99%

Endogenous, tissue-resident stem/progenitor cells in gonads and bone marrow express FSHR and respond to FSH via FSHR-3

et al. 2021

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Follicle stimulating hormone (FSH) is secreted by the anterior pituitary and acts on the germ cells indirectly through Granulosa cells in ovaries and Sertoli cells in the testes. Extragonadal action of FSH has been reported but is still debated. Adult tissues harbor two populations of stem cells including a reserve population of primitive, small-sized, pluripotent very small embryonic-like stem cells (VSELs) and slightly bigger, tissue-specific progenitors which include ovarian stem cells (OSCs) in ovaries, spermatogonial stem cells (SSCs) in testes, endometrial stem cells (EnSCs) in uterus and hematopoietic stem cells (HSCs) in the bone marrow. Data has accumulated in animal models showing FSHR expression on both VSELs and progenitors in ovaries, testes, uterus and bone marrow and eventually gets lost as the cells differentiate further. FSH exerts a direct action on the stem/progenitor cells via alternatively spliced FSHR-3 rather than the canonical FSHR-1. FSH stimulates VSELs to undergo asymmetrical cell divisions to self-renew and give rise to the progenitors that in turn undergo symmetrical cell divisions and clonal expansions followed by differentiation into specific cell types. Excessive self-renewal of VSELs results in cancer and this explains ubiquitous expression of embryonic markers including nuclear OCT-4 along with FSHR in cancerous tissues. Focus of this review is to compile published data to support this concept. FSHR expression in stem/progenitor cells was confirmed by immuno-fluorescence, Western blotting, in situ hybridization and by quantitative RT-PCR. Two different commercially available antibodies (Abcam, Santacruz) were used to confirm specificity of FSHR expression along with omission of primary antibody and pre-incubation of antibody with immunizing peptide as negative controls. Western blotting allowed detection of alternatively spliced FSHR isoforms. Oligoprobes and primers specific for Fshr-1 and Fshr-3 were used to study these alternately-sliced isoforms by in situ hybridization and their differential expression upon FSH treatment by qRT-PCR. To conclude, stem/progenitor cells in adult tissues express FSHR and directly respond to FSH via FSHR-3. These findings change the field of FSH-FSHR biology, call for paradigm shift, explain FSHR expression on cancer cells in multiple organs and provide straightforward explanations for various existing conundrums including extragonadal expression of FSHR.

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