Very small embryonic-like stem cells (VSELs) are possibly lost during cord blood banking and bone marrow (BM) processing for autologus stem cell therapy mainly because of their small size. The present study was conducted on human umbilical cord blood (UCB, n=6) and discarded red blood cells (RBC) fraction obtained after separation of mononuclear cells from human BM (n=6), to test this hypothesis. The results show that VSELs, which are pluripotent stem cells with maximum regenerative potential, settle along with the RBCs during Ficoll-Hypaque density separation. These cells are very small in size (3-5 μm), have high nucleo-cytoplasmic ratio, and express nuclear Oct-4, cell surface protein SSEA-4, and other pluripotent markers such as Nanog, Sox-2, Rex-1, and Tert as indicated by immunolocalization and quantitative polymerase chain reaction (Q-PCR) studies. Interestingly, a distinct population of slightly larger, round hematopoietic stem cells (HSCs) with cytoplasmic Oct-4 were detected in the "buffy" coat, which usually gets banked or used during autologus stem cell therapy. Immunohistochemical studies on the umbilical cord tissue (UCT) sections (n=3) showed the presence of nuclear Oct-4-positive VSELs and many fibroblast-like mesenchymal stem cells (MSCs) with cytoplasmic Oct-4. These VSELs with nuclear Oct-4, detected in UCB, UCT, and discarded RBC fraction obtained after BM processing, may persist throughout life, maintain tissue homeostasis, and undergo asymmetric cell division to self-renew as well as produce larger progenitor stem cells, viz. HSCs or MSCs, which follow differentiation trajectories depending on the somatic niche. Hence, it can be concluded that the true stem cells in adult body tissues are the VSELs, whereas the HSCs and MSCs are actually progenitor stem cells that arise by asymmetric cell division of VSELs. The results of the present study may help explain low efficacy reported during adult autologous stem cell trials, wherein unknowingly progenitor stem cells are injected rather than the pluripotent stem cells with maximum regenerative potential.
Being quiescent, VSELs possibly do not accumulate genomic (nuclear or mitochondrial) mutations and thus may be ideal endogenous, pluripotent stem cell candidates for regenerative and reproductive medicine. The presence of VSELs in adult gonads and the fact that they survive oncotherapy may obviate the need to bank gonadal tissue for fertility preservation prior to oncotherapy. VSELs and their ability to undergo spermatogenesis/neo-oogenesis in the presence of a healthy niche will help identify newer strategies toward fertility restoration in cancer survivors, delaying menopause and also enabling aged mothers to have better quality eggs.
It has been suggested that testicular germ stem cells represent the only adult body stem cells that dedifferentiate and reprogram into a pluripotent state without any genetic modification. Emerging debate about the authenticity of embryonic stem cell (ES)-like cells derived from adult testicular tissue has prompted us to put forth this letter. We wish to reinforce our findings that pluripotent very small embryonic-like stem cells (VSELs) exist as a small population in adult mammalian testis and may result in ES-like colonies. Because of their small size, it is felt that VSELs could be contaminating the initial cells used for seeding, although efforts were made to place a single germ cell per well in a 96-well plate for clonal expansion, or magnetic activated cell sorting (MACS)-sorted α6 integrin positive cells were used. On a similar note, it is felt that the presence of VSELs in various tissues along with mesenchymal stem cells (MSCs) may provide an alternative explanation to the transdifferentiation potential of MSCs. We conclude that like Oct-4 biology, presence of VSELs in adult body tissues has somewhat surprised stem cell biologists.
Infectious respiratory particles expelled by SARS-CoV-2 positive patients are attributed to be the key driver of COVID-19 transmission. Understanding how and by whom the virus is transmitted can help implement better disease control strategies. Here we have described the use of a noninvasive mask sampling method to detect and quantify SARS-CoV-2 RNA in respiratory particles expelled by COVID-19 patients and discussed its relationship to transmission risk. Respiratory particles of 31 symptomatic SARS-CoV-2 positive patients and 31 asymptomatic healthy volunteers were captured on N-95 masks layered with a gelatin membrane in a 30-minute process that involved talking/reading, coughing, and tidal breathing. SARS-CoV-2 viral RNA was detected and quantified using rRT-PCR in the mask and in concomitantly collected nasopharyngeal swab (NPS) samples. The data were analyzed with respect to patient demographics and clinical presentation. Thirteen of 31(41.9%) patients showed SARS-COV-2 positivity in both the mask and NPS samples, while 16 patients were mask negative but NPS positive. Two patients were both mask and NPS negative. All healthy volunteers except one were mask and NPS negative. The mask positive patients had significantly lower NPS Ct value (26) compared to mask negative patients (30.5) and were more likely to be rapid antigen test positive. The mask positive patients could be further grouped into low emitters (expelling <100 viral copies) and high emitters (expelling >1000 viral copies). The study presents evidence for variation in emission of SARS-CoV-2 virus particles by COVID-19 patients reflecting differences in infectivity and transmission risk among individuals. The results conform to reported secondary infection rates and transmission and also suggest that mask sampling could be explored as an effective tool to assess individual transmission risks, at different time points and during different activities.
BackgroundPluripotent, Lin–/CD45–/Sca-1+ very small embryonic-like stem cells (VSELs) in mouse bone marrow (BM) are resistant to total body radiation because of their quiescent nature, whereas Lin–/CD45+/Sca-1+ hematopoietic stem cells (HSCs) get eliminated. In the present study, we provide further evidence for the existence of VSELs in mouse BM and have also examined the effects of a chemotherapeutic agent (5-fluorouracil (5-FU)) and gonadotropin hormone (follicle-stimulating hormone (FSH)) on BM stem/progenitor cells.MethodsVSELs and HSCs were characterized in intact BM. Swiss mice were injected with 5-FU (150 mg/kg) and sacrificed on 2, 4, and 10 days (D2, D4, and D10) post treatment to examine changes in BM histology and effects on VSELs and HSCs by a multiparametric approach. The effect of FSH (5 IU) administered 48 h after 5-FU treatment was also studied. Bromodeoxyuridine (BrdU) incorporation, cell cycle analysis, and colony-forming unit (CFU) assay were carried out to understand the functional potential of stem/progenitor cells towards regeneration of chemoablated marrow.ResultsNuclear OCT-4, SCA-1, and SSEA-1 coexpressing LIN–/CD45– VSELs and slightly larger LIN–/CD45+ HSCs expressing cytoplasmic OCT-4 were identified and comprised 0.022 ± 0.002 % and 0.081 ± 0.004 % respectively of the total cells in BM. 5-FU treatment resulted in depletion of cells with a 7-fold reduction by D4 and normal hematopoiesis was re-established by D10. Nuclear OCT-4 and PCNA-positive VSELs were detected in chemoablated bone sections near the endosteal region. VSELs remained unaffected by 5-FU on D2 and increased on D4, whereas HSCs showed a marked reduction in numbers on D2 and later increased along with the corresponding increase in BrdU uptake and upregulation of specific transcripts (Oct-4A, Oct-4, Sca-1, Nanog, Stella, Fragilis, Pcna). Cells that survived 5-FU formed colonies in vitro. Both VSELs and HSCs expressed FSH receptors and FSH treatment enhanced hematopoietic recovery by 72 h.ConclusionBoth VSELs and HSCs were activated in response to the stress created by 5-FU and FSH enhanced hematopoietic recovery by at least 72 h in 5-FU-treated mice. VSELs are the most primitive pluripotent stem cells in BM that self-renew and give rise to HSCs under stress, and HSCs further divide rapidly and differentiate to maintain homeostasis. The study provides a novel insight into basic hematopoiesis and has clinical relevance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0311-6) contains supplementary material, which is available to authorized users.
Very small embryonic-like stem cells (VSELs) are immature primitive cells residing in adult and fetal tissues. This study describes enrichment strategy and molecular and phenotypic characterization of human cord blood VSELs. Flow cytometry analysis revealed that a majority of VSELs (LIN(-)/CD45(-)/CD34(+)) were present in the red blood cell (RBC) pellet after Ficoll-Hypaque centrifugation in contrast to the hematopoietic stem cells (LIN(-)/CD45(+)/CD34(+)) in the interphase layer. Thus, after lyses of RBCs, VSELs were enriched using CD133 and SSEA4 antibodies. These enriched cells were small in size (4-6 μm), spherical, exhibited telomerase activity and expressed pluripotent stem cell (OCT4A, OCT4, SSEA4, NANOG, SOX2, REX1), primordial germ cell (STELLA, FRAGILIS) as well as primitive hematopoietic (CD133, CD34) markers at protein and transcript levels. Heterogeneity was noted among VSELs based on subtle differences in expression of various markers studied. DNA analysis and cell cycle studies revealed that a majority of enriched VSELs were diploid, non-apoptotic and in G0/G1 phase, reflecting their quiescent state. VSELs also survived 5-fluorouracil treatment in vitro and treated cells entered into cell cycle. This study provides further support for the existence of pluripotent, diploid and relatively quiescent VSELs in cord blood and suggests further exploration of the subpopulations among them.
Adult tissues are thought to harbor two populations of "dormant" and "actively dividing" stem cells. Quiescent stem cells undergo rare asymmetric cell divisions (ACDs) through which they self-renew and give rise to tissue-committed "progenitors" of distinct fate and "progenitors" in turn undergo symmetric cell divisions (SCDs) and clonal expansion. However, quiescent stem cells have not been demonstrated in adult tissues such as skin, testis, liver, and brain. After surgical removal of part of liver and pancreas-adult differentiated cells divide and regenerate and a possible role of stem cells remains doubtful. Long-term repopulating hematopoietic stem cells are quiescent in nature but ACD has not been convincingly demonstrated even among them. Attempts by various groups to identify a common stemness program that ensures self-renewal among different kinds of stem cells have also remained futile. Uncontrolled self-renewal and compromised differentiation of stem cells possibly initiate leukemia/cancer, but the identity of leukemic stem cells and whether cancer stem cells arise by epithelial-mesenchymal transition (EMT) in solid tumors are all open-ended questions that need greater clarity. Acceptance of the presence of very small embryonic-like stem cells (VSELs) in adult tissues could clarify several of these existing dilemmas in the field. Data are compiled showing that VSELs undergo ACD in the hematopoietic system, testis, ovary, uterus, and pancreas, whereas tissue-committed progenitors undergo SCD and clonal expansion. VSELs possess similar overlapping stemness program as in embryonic stem cells, embryonic carcinoma cells, embryonic germ cells, induced pluripotent stem cells, and primordial germ cells. VSELs and leukemic and cancer cells express overlapping embryonic markers. Uncontrolled proliferation of VSELs and compromised differentiation possibly initiate leukemia. Process of EMT and initiation of solid tumor from VSELs (located among the epithelial cells) are indeed two distinct and parallel events. To conclude, VSELs provide explanation to several confounding aspects of adult stem cell biology.
Very small embryonic-like stem cells (VSELs) have been reported in various adult tissues, express pluripotent and primordial germ cells (PGCs) specific markers, are mobilized under stress/disease conditions, give rise to tissue committed progenitors and thus help regenerate and maintain homeostasis. The aim of the present study was to evaluate in vitro differentiation potential of VSELs using a quantitative approach. VSELs were collected from mouse bone marrow after 4 days of 5-fluorouracil (5-FU, 150 mg/Kg) treatment, further enriched by size based filtration and cultured on a feeder support in the presence of specific differentiation media. Cultured VSELs were found to differentiate into all three embryonic germ cell lineages, germ and hematopoietic cells after 14 days in culture. This was confirmed by studying Nestin, PDX-1, NKX2.5, DAZL, CD45 and other markers expression by various approaches. Very small, CD45 negative cells collected and enriched from GFP positive 5-FU treated mice bone marrow transitioned into CD45 positive cells in vitro thus demonstrating that VSELs can give rise to hematopoietic stem cells (HSCs). We envision that VSELs may be responsible for plasticity and ability of bone marrow cells to give rise to non-hematopoietic tissue progenitors of all 3 germ layers. Moreover the ability of VSELs to differentiate into germ cells as well as all the three lineages provides further evidence to support their pluripotent state and confirms developmental link between bone marrow VSELs and PGCs. The property of quiescence, no risk of teratoma formation and autologus source, make pluripotent VSELs a potential candidate to facilitate endogenous regeneration compared to cell replacement strategy envisioned using embryonic and induced pluripotent stem cells.
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