A Novel View of the Adult Stem Cell Compartment From the Perspective of a Quiescent Population of Very Small Embryonic-Like Stem Cells

Abstract

Evidence has accumulated that adult hematopoietic tissues and other organs contain a population of dormant stem cells (SCs) that are more primitive than other, already restricted, monopotent tissue-committed SCs (TCSCs). These observations raise several questions, such as the developmental origin of these cells, their true pluripotent or multipotent nature, which surface markers they express, how they can be efficiently isolated from adult tissues, and what role they play in the adult organism. The phenotype of these cells and expression of some genes characteristic of embryonic SCs, epiblast SCs, and primordial germ cells suggests their early-embryonic deposition in developing tissues as precursors of adult SCs. In this review, we will critically discuss all these questions and the concept that small dormant SCs related to migratory primordial germ cells, described as very small embryonic-like SCs, are deposited during embryogenesis in bone marrow and other organs as a backup population for adult tissue-committed SCs and are involved in several processes related to tissue or organ rejuvenation, aging, and cancerogenesis. The most recent results on successful ex vivo expansion of human very small embryonic-like SC in chemically defined media free from feeder-layer cells open up new and exciting possibilities for their application in regenerative medicine.

Keywords: adult stem cells; germ layers; hematopoietic stem cells; parental imprinting; ras-GRF1; stem cells; very small embryonic like stem cells.

Figure 1. Identification and isolation of VSELs and HSCs from human umbilical cord blood by employing FACS sorter

To develop a more efficient and less time consuming method for purifying VSELs from UCB, we developed a three-step isolation strategy based on (1) removing erythrocytes by hypotonic lysis, (2) immunomagnetic separation of CD133⁺ cells, and (3) FACS-based isolation of small CD133⁺Lin⁻CD45⁻ cells. Region R1 shows events exhibiting DNA content. Nucleated cells included in R2 are visualized based on their FSC and SSC characteristics. R3 and R4 are employed to exclude doublets. Region R5 presents Lin⁻ cells, which are analyzed on next dot-plot based on CD133 vs. CD45 expression and clasified as VSELs enclosed in R7 (CD133⁺Lin⁻CD45⁻), and HSCs enclosed in R6 (CD133⁺Lin⁻CD45⁺). Small and agranular VSELs from region R7 are presented on cytogram based on FSC vs. SSC characteristics (green ellipse), compared to bigger HSCs from region R6 (blue ellipse).

Figure 2. Example of expansion of human umbilical cord blood derived VSELs

Panel A – Freshly sorted VSELs (5×10²) were plated in 0.2 ml of DMEM + 10% FBS, supplemented with VPA and a cocktail of two pituitary sex hormones, FSH and LH together with BMP-4, IGF-2 and KL. Right inset shows enlarged image of freshly sorted VSEL. Cells were cultured for 2 months and half of culture medium has been changed every 7 days. Panel B – Upper panel - VSELs in these culture conditions began to proliferate, and after 2 months of expansion we can distinguish many small cells as well as some larger cells. Maximal expansion is achieved after 2–3 months of culture. Lower panel - cells aspirated from the cultures. Left and middle panel light microscope image. Right panel – Hoe3342 intravital staining of cells aspirated from the expansion.