GFP Tagged VSELs Help Delineate Novel Stem Cells Biology in Multiple Adult Tissues.

Various types of stem cells are being researched upon to exploit their potential for regenerative medicine including pluripotent human embryonic stem (hES) cells derived from spare human embryos, induced pluripotent stem (iPS) cells by reprogramming somatic cells to a pluripotent state and multipotent mesenchymal stem/stromal cells (MSCs) obtained in vitro from multiple tissues. More than 50 independent groups have reported another novel population of pluripotent stem cells in adult tissues termed very small embryonic-like stem cells (VSELs). VSELs are developmentally linked to primordial germ cells, which rather than giving rise to the germ cells and later ceasing to exist, survive throughout life in multiple organs along with tissue-specific adult stem cells better described as lineage-restricted, tissue-committed progenitors with limited plasticity. VSELs survive total body irradiation in bone marrow, oncotherapy in the gonads, bilateral ovariectomy in the uterus and partial pancreatectomy in the pancreas of mice and participate in the regeneration of multiple organs under normal physiological conditions. VSELs and tissue-specific progenitor cells work together in a subtle manner, maintain life-long tissue homeostasis and their dysfunction leads to various pathologies including cancer. However, due to their quiescent state, VSELs have invariably eluded lineage-tracing studies reported so far. Present article reviews novel insights into VSELs biology and how VSELs enriched from GFP (green fluorescent protein) mice have enabled to delineate their role in various biological processes in vivo. VSELs biology needs to be understood in-depth as this alone will help evolve the field of regenerative medicine and win the war against cancer.

Two Stem Cell Populations Including VSELs and CSCs Detected in the Pericardium of Adult Mouse Heart.

Adult mammalian heart is considered to be one of the least regenerative organs as it is not able to initiate endogenous regeneration in response to injury unlike in lower vertebrates and neonatal mammals. Evidence is now accumulating to suggest normal renewal and replacement of cardiomyocytes occurs even in middle-aged and old individuals. But underlying mechanisms leading to this are not yet clear. Do tissue-resident stem cells exist or somatic cells dedifferentiate leading to regeneration? Lot of attention is currently being focused on epicardium as it is involved in cardiac development, lodges multipotent progenitors and is a source of growth factors. Present study was undertaken to study the presence of stem cells in the pericardium. Intact adult mouse heart was subjected to partial enzymatic digestion to collect the pericardial cells dislodged from the surface. Pericardial cells suspension was processed to enrich the stem cells using our recently published protocol. Two populations of stem cells were successfully enriched from the pericardium of adult mouse heart along with distinct 'cardiospheres' with cytoplasmic continuity (formed by rapid proliferation and incomplete cytokinesis). These included very small embryonic-like stem cells (VSELs) and slightly bigger 'progenitors' cardiac stem cells (CSCs). Expression of pluripotent (Oct-4A, Sox-2, Nanog), primordial germ cells (Stella, Fragilis) and CSCs (Oct-4, Sca-1) specific transcripts was studied by RT-PCR. Stem cells expressed OCT-4, NANOG, SSEA-1, SCA-1 and c-KIT. c-KIT was expressed by cells of different sizes but only smaller CD45-c-KIT+ VSELs possess regenerative potential. Inadvertent loss of stem cells while processing for different experiments has led to misperceptions & controversies existing in the field of cardiac stem cells and requires urgent rectification. VSELs/CSCs have the potential to regenerate damaged cardiac tissue in the presence of paracrine support provided by the mesenchymal stromal cells.

Which stem cells will eventually translate to the clinics for treatment of diabetes?

Human embryonic stem (hES) cells have been around for more than two decades now. It was expected that hES/iPS (induced pluripotent stem) cells will quickly translate to the clinics to treat diabetic patients and to obtain gametes in vitro for infertile couples. However, there is no breakthrough yet in either of the fields although considerable progress has been made. Research efforts are ongoing to obtain an insight into the gene expression changes associated with directed differentiation of hES/iPS cells. Autologous bone marrow/cord blood mononuclear cells' therapy has also failed to show any regenerative potential and only remains as a standard method of care for blood diseases. Only mesenchymal stem cells (MSCs) have shown promise in the clinics to alleviate diabetic symptoms. But MSCs are stromal cells with no regenerative properties; rather "paracrine providers", pericytes/stromal cells, better known for their trophic, immuno-modulatory, and anti-inflammatory properties and thus best termed as mesenchymal stromal cells (MSCs). Autologus bone marrow cells enriched for hematopoietic stem cells have no potential to cross boundaries and transdifferentiate into other lineages including endodermal pancreatic cells. Endogenous, pluripotent, very small embryonic-like stem cells (VSELs) emerge as the most likely endogenous stem cell candidates to regenerate adult diabetic pancreas. Transplanted MSCs provide a healthy paracrine support required for endogenous/ resident VSELs to differentiate into acinar cells and islets in a diabetic pancreas to enable restoration of homeostasis. Our recently published study shows that VSELs exist and can be enriched from intact mouse pancreas as well as from the islets and increase in numbers in diabetic pancreas. Providing "regenerative pressure" by subjecting diabetic mice to partial pancreatectomy stimulated the VSELs to undergo differentiation into various cell types in an attempt to restore homeostasis. Double-blinded, placebo controlled clinical trials need to be undertaken to evaluate the efficacy of transplanting MSCs in diabetic patients with conviction since now underlying fine play of endogenous VSELs and niche providing MSCs has emerged.

Mouse Pancreas Stem/Progenitor Cells Get Augmented by Streptozotocin and Regenerate Diabetic Pancreas After Partial Pancreatectomy.

Existence of stem cells in adult pancreas remains contentious. Single cells suspensions obtained by collagenase and trypsin digestion separately from adult mouse pancreas and pancreatic islets were spun at 1000 rpm (250 g) to collect the cells. At this speed the stem/ progenitor cells remained buoyant and were further enriched by spinning the supernatant at 3000 rpm (1000 g). Two distinct populations of stem cells were detected including pluripotent, very small (2-6 µm) embryonic-like stem cells (VSELs) that expressed nuclear OCT-4A and pluripotent transcripts (Oct-4A, Sox2, Nanog, Stella) and slightly bigger progenitors, pancreatic stem cells (PSCs) that expressed cytoplasmic OCT-4B and PDX-1. Streptozotocin treated diabetic pancreas showed an increase in numbers of VSELs (2-6 µm, 7AAD-, LIN-CD45-SCA1+ cells) and up-regulation of transcripts specific for stem/ progenitor cells. Diabetic mice were further subjected to partial pancreatectomy to study involvement of VSELs/ PSCs during regeneration. VSELs/ PSCs were mobilized in large numbers, were observed in the lumen of blood vessels and PCNA expression suggested their proliferation. Initially, new acini assembled to regenerate the exocrine pancreas and later by Day 30, neogenesis of islets was observed in the vicinity of the blood vessels and pancreatic ducts by the differentiation of endogenous VSELs/ PSCs which may be targeted to regenerate diabetic pancreas in clinical settings.