Recently, rare CXC chemokine receptor-4-expressing (CXCR4 ⁺ ) small stem cells that express markers characteristic for embryonic stem cells, epiblast stem cells and primordial germ cells were purified from murine bone marrow. Based on the morphology of these cells and unique molecular signature of gene expression, this primitive cell population was named “very small embryonic-like (VSEL) stem cells”. The data indicate that VSELs are also present in many other murine organs, including kidney, and that they may differentiate into cells from all three germ layers. It is hypothesized that VSELs are deposited during gastrulation and organogenesis in developing mammalian organs/tissues (e.g. kidney) as a population of pluripotent stem cells that give rise to monopotent tissue-committed stem cells and that their number decreases during aging. Based on this, VSELs could play a pivotal role in normal rejuvenation of adult tissues as well as being involved in the regeneration of damaged organs. It is hypothesized that VSELs originate from epiblast/migrating primordial germ cell-like cells and, despite expression of pluripotent stem cell markers, changes in the epigenetic signature of imprinted genes keep these cells quiescent in adult tissues. Perturbation in expression of imprinted genes in VSELs deposited in kidney (e.g. on Igf2-H191 and Kcnq1 loci) may give rise to Wilms’ tumor, as seen for example in Beckwith–Wiedemann syndrome. Finally, the controlled modulation of somatic imprint status in VSELs, such as proper modulation of somatic imprinted genes, could increase the regenerative power of these cells. Thus, VSELs are promising cell candidates for regenerative medicine and could be harnessed to regenerate damaged tissues and decelerate the aging processes. Since similar cells are also present in human cord blood and human mobilized peripheral blood, it may be assumed that they also reside in human kidneys.

Introduction

Continuous tissue and organ regeneration is one of the important homeostatic mechanisms of the multicellular organism. The senescent, functionally differentiated mature cells in adult organs are continuously being replaced by new ones . This cell turnover is more rapid in the hematopoietic system, intestinal epithelium and epidermis than in the kidney, skeletal muscles, liver and heart. Old or senescent cells in adult organisms are eliminated by various mechanisms involving: apoptosis (e.g. neurons), shedding from the epithelial and epidermal surfaces (e.g. enterocytes, keratinocytes) or active elimination by phagocytosis (e.g. senescent erythrocytes). Thus, to keep a cell mass balance in different tissues and organs, new cells have to be generated continuously to replace eliminated ones . The new cells are supplied by a pool of tissue-committed stem cells (TCSCs) that reside in specific niches spread throughout the organs and tissues . In the kidney, for example, the presence of the so-called renopoietic system was postulated with TCSCs localized at the urinary pole of Bowman’s capsule, from where they can initiate the replacement and regeneration of glomerular, as well as tubular, epithelial cells .

In general, when dividing, stem cells have three choices ( Fig. 12.1 A): they give rise to two daughter stem cells (symmetric division), one stem cell and a differentiated progenitor cell (asymmetric division), or two progenitor cells. This selection of the stem cell fate is a randomly determined process, which follows a probability distribution that can be analyzed statistically, but cannot be determined precisely (stochastic selection).

Stem cell contribution to tissue rejuvenation. (A) Stem cells can give rise to: (a) two daughter stem cells (symmetric division); (b) one stem cell and differentiated progenitor cell (asymmetric division); and (c) two progenitor cells. (B) Pluripotent stem cells that are deposited in adult tissues during embryogenesis/gastrulation may become eliminated after giving rise to tissue-committed stem cells (TCSCs) or hypothetically survive among TCSCs and serve as a potential reserve source of TCSCs.

As mentioned above, while some tissues regenerate quickly, others regenerate at a very slow pace. For example, turnover of the intestinal epithelium takes place every 48 h, the epidermis is exchanged every 14 days, and the half-life of erythrocytes circulating in peripheral blood is 100–150 days. New evidence indicates that even organs such as the kidney, heart or brain also show some degree of cellular turnover, although that occurs very slowly . In the case of the kidney, recent evidence shows that even podocytes, a cell type with limited proliferative capacity under normal conditions, are constantly regenerated from a population of renopoietic cells within Bowman’s capsule. To support this, Romagnani and co-workers identified in this region a population of renal CD133 ⁺ CD24 ⁺ multipotent progenitors that have the capacity for self-renewal and multilineage differentiation . The authors postulated that these cells represent a subset of multipotent embryonic stem/progenitor cells that persist in human kidneys from the early stages of embryonic nephrogenesis.

The novel hypothetical concept presented in this chapter addresses data that support the residence of some very primitive pluripotent stem cells (PSCs) in adult tissues with the ability to differentiate into multiple types of TCSCs. In addition to above-mentioned CD133 ⁺ CD24 ⁺ multipotent progenitors residing in Bowman’s capsule, several investigators have described cells in adult tissues with remarkably broad spectra of differentiation. These cells were described in the literature as multipotent adult progenitor cells (MAPCs), marrow-isolated adult multilineage inducible (MIAMI) cells, multipotent adult stem cells (MASCs) or OmniCytes . Recently, the authors’ team purified from murine bone marrow (BM) rare CXCR4 ⁺ small stem cells that express markers characteristic for embryonic stem cells (ESCs), epiblast stem cells (EpiSCs) and primordial germ cells (PGCs) . Based on the morphology of these cells and unique molecular signature of gene expression, these primitive cells were named very small embryonic-like (VSEL) stem cells.

It cannot be excluded that similar or overlapping populations of primitive stem cells in adult tissues were detected using various experimental strategies and, subsequently, were assigned different names. However, it is an important challenge to understand the biological significance of these primitive stem cells residing in adult tissues. Are these cells merely developmental remnants that are deposited during embryogenesis in the adult tissues, or do they serve as a reserve pool for TCSCs ( Fig. 12.1 B)? If the latter possibility is true, another question emerges: are they continuously supplying and rejuvenating a pool of TCSCs or are these cells a source of TCSCs in emergency situations only, as seen in tissue or organ injury (e.g. kidney damage)? Finally, it is important to explore in the future the relationship between VSELs and TCSCs from the renopoietic system located in Bowman’s capsule.

Developmental Overview on Stem Cell Compartment

It is well known that during embryogenesis the stem cell compartment is heterogeneous and that early stem cells are endowed with a remarkable potential to differentiate into tissues from one, two or even three germ layers . The fertilized oocyte or zygote is unquestionably the most primitive stem cell from which a whole organism develops. The zygote is a totipotent stem cell, the most primitive stem cell which gives rise to both placenta and embryo proper . In a relatively short time, just a few days, the zygote gives rise to the morula and blastocyst. Stem cells in the morula and inner cell mass of the blastocyst are pluripotent. PSCs give rise to TCSCs from all three germ layers (mesoderm, ectoderm and endoderm) that form the embryo proper.

The development of an embryo into an adult organism during embryogenesis is subsequently regulated by the coordinated proliferation, specification and differentiation of TCSCs . As a result of this, one fertilized oocyte gives rise to an adult organism that consists of more than 200 different cell types which total, in the case of humans approximately 10 ¹⁴ somatic cells. In adult organisms, various types of TCSCs reside in specific niches (e.g. Bowman’s capsule in kidney, bottom of intestinal crypts, hair bulge) and replace senescent cells within various organs . It is also accepted that monopotent TCSCs are endowed with the property of self-renewal and the ability to differentiate into progenitor cells that are committed to particular developmental pathways. Since, as discussed above, somatic cells have a limited half-life, continuous tissue and organ regeneration by TCSCs is one of the important homeostatic mechanisms that maintain the homeostasis of the multicellular organism .

Deposition of Developmentally Early Stem Cells in Adult Tissues

Reports that monopotent TCSCs are plastic and can change differentiation commitment (trans-dedifferentiate) into stem cells for other tissues are controversial . However, the presence of pluripotent or multipotent stem cells in adult tissues may much better explain some of the positive results demonstrating data on the plasticity of stem cells. Evidence shows that in addition to TCSCs, adult organs harbor a population of more primitive PSCs, that may be envisioned as a reserve stem cells population for TCSCs. As mentioned above, several groups including the present authors have reported on the presence of developmentally primitive stem cell populations with PSC properties that are distributed in adult organs and tissues (e.g. MAPCs, MIAMI cells, MASCs, OmniCytes and VSELs).

These cells reside in adult tissues and could be activated or attracted during stress or tissue injuries to regenerate damaged organs . It is well known that damaged organs secrete several factors such as stromal-derived factor-1/CXC chemokine ligand-12 (SDF-1)/(CXCL-12), hepatocyte growth factor/scatter factor (HGF/SF), leukemia inhibitor factor (LIF), and vascular endothelial growth factor (VEGF) which may chemoattract several of the primitive stem cell populations . In support of this, damaged tissues [e.g. kidney during acute renal failure (ARF), infarcted myocardium or stroke area] secrete several of these factors during hypoxia (deprivation of adequate oxygen supply) . The most important ones are products of genes regulated at the transcriptional level by a transcription factor called hypoxia-inducible factor-1α (HIF-1α). Accordingly, HIF-1α regulates several genes including SDF-1α/CXCL12, HGF/SF and VEGF . All these factors (SDF-1, HGF/SF and VEGF) orchestrate accumulation of stem cells in damaged tissues, e.g. myocardium after infarction, SDF-1 interacts with CXCR4 and, as recently reported, with another seven transmembrane-span G-protein-coupled CXC chemokine receptor 7 (CXCR7) as well . It has been demonstrated that SDF-1–CXCR4 and SDF-1–CXCR7 axes play an important role in trafficking of renal progenitors as evidenced, for example, in experimental ARF in mice . To support this notion, in severe combined immunodeficiency (SCID) mice with ARF, SDF-1 is strongly upregulated in cells that surround necrotic areas. Furthermore, intravenously injected renal CXCR4 ⁺ CXCR7 ⁺ TCSCs engrafted into injured SDF-1-enriched renal tissue both decreased the severity of ARF and prevented renal fibrosis.

Other important players in the trafficking of stem cells to the damaged tissues are bioactive peptides released during complement cascade (CC) activation. The complement system, which is a part of the body’s innate immunity, also becomes activated in injured and hypoxic tissues . It has been reported that the most abundant product of complement cleavage/activation is the small fragment of the third complement component 3 (C3) called C3a, which increases and sensitizes the responsiveness of stem cells to SDF-1 gradient . Recently, other factors were identified from the small cationic peptides family (e.g. cathelicidin, β-II-defensin), which are secreted by granulocytes that accumulate in damaged tissues and, similarly to C3a, increase the responsiveness of TCSCs to the SDF-1 gradient . Thus, in addition to SDF-1/CXCL12 and growth factors, CC cleavage fragments have an important role in the recruitment of stem cells to the damaged tissues, e.g. kidney ( Fig. 12.2 ). However, the interplay of SDF-1 and C3a in kidney regeneration during ARF requires further study. It is also important to assess a potential role of VSELs in this process.

Very small embryonic-like stem cells (VSELs) are mobilized into peripheral blood, e.g. in kidney damage. (A) Under normal steady state conditions, VSELs may circulate in peripheral blood to keep a pool of stem cells in balance in distant niches of the same tissue. (B) The number of these cells increases during stress related to organ/tissue damage. During organ damage (e.g. hypoxic damage of kidney), the level of stromal derived factor-1 (SDF-1) is upregulated in the affected tissues and C3 becomes activated, leading to the accumulation of C3 cleavage fragments (C3a and _desArg C3a). C3 cleavage fragments enhance/prime the responsiveness of circulating CXCR4 ⁺ stem cells to an SDF-1 gradient. This leads to more efficient chemoattraction of stem cells for potential regeneration of the damaged tissue by creating a supergradient, as shown in (B) for damaged kidney, for example. In addition to SDF-1, other chemoattractants play important roles here (e.g. hepatocyte growth factor/scatter factor, leukemia inhibitory factor and vascular endothelial growth factor).

To support this hypothesis, it has been reported that PSCs (e.g. VSELs or OmniCytes) could be mobilized into peripheral blood during several models of organ injury and circulate there in an attempt to enrich and regenerate damaged tissues . This physiological mechanism probably plays a more significant role in the regeneration of some small tissue and organ injuries. The regeneration of major tissue or organ damage will require local delivery of a higher number of pluripotent or multipotent stem cells. In general, the regeneration mechanisms are more efficient at a young age, which could be explained by the number of these primitive mobile stem cells (e.g. VSELs) decreasing with age . Thus, this aging-related decrease in the number of VSELs could explain both decreasing rejuvenation potential and impaired organ regeneration with advanced age.

Isolation of Very Small Embryonic-like Stem Cells

Using fluorescence-activated cell sorting (FACS), a homogeneous population of rare small Sca-1 ⁺ /Lin ⁻ /CD45 ⁻ cells has been isolated from BM as well from several other adult tissues including brain, liver, pancreas, kidney, muscles, heart, testes and thymus . These VSELs, as determined by real-time quantification reverse transcription polymerase chain reaction (RT-PCR) and by immunohistochemistry, express several markers of PSCs such as SSEA-1, Oct-4, Nanog, and Rex-1, as well as Rif-1 telomerase protein.

To isolate VSELs from the BM by FACS, a novel size-based approach controlled by size bead markers was used. The overall sorting strategy was to gate in regions containing small events (2–10 μm), shown as region R1 on the dot plot ( Fig. 12.3 ). This region mostly contains cell debris, but also has some rare nucleated cell events. Because most of the sorting protocols exclude events smaller than 6 μm in diameter that contain cell debris, erythrocytes and platelets, these small VSELs are usually excluded from the sorted cell populations. In this sorting strategy for VSELs, the size of the sorted cells is controlled by the beads with predefined sizes (1, 2, 4, 6, 10 and 15 μm in diameter).

Sorting strategy for isolation of murine bone marrow-derived very small embryonic-like stem cells (VSELs) by FACS. Bone marrow-derived VSELs were sorted by MoFlo cell sorter (Dako, Glostrup, Denmark) following immunofluorescence staining for Sca-1, CD45 and hematopoietic Lin. (A) Distribution of six predefined, sized bead populations according to their forward and side scatter characteristics (FSCs and SSCs, respectively). Gate R1 includes objects between 2 and 10 μm in size after comparison to bead particles with standard sizes of 1, 2, 4, 6, 10 and 15 μm (Flow Cytometry Size beads, Invitrogen; Molecular Probes, Carlsbad, CA, USA). (B) Bone marrow mononuclear cells visualized on dot plots showing their FSC and SSC signals related to the size and granularity/complexity of the cell, respectively. Small, agranular cells included in region R1 are further visualized based on the expression of Sca-1 and Lin markers (D). Region R2 includes only Sca-1 ⁺ /Lin ⁻ , which are subsequently sorted based on CD45 marker expression into CD45 ⁻ and CD45 ⁺ subpopulations visualized on a histogram (C). Sca-1 ⁺ /Lin ⁻ /CD45 ⁻ cells (VSELs) are sorted as events enclosed in a logical gate including regions R1, R2 and R3, while Sca-1 ⁺ /Lin ⁻ /CD45 ⁺ cells (hematopoietic stem cells) from the gate include regions R1, R2 and R4. Approximate percent contents of each cellular subpopulation are indicated on the plots.

The events enclosed in region R1 ( Fig. 12.3 A, B), which include an average of ~ 50% of total events, are further analyzed for the expression of Sca-1 and lineage markers (Lin). The Sca-1 ⁺ /Lin ⁻ events shown in region R2 ( Fig. 12.3 D) consist of 0.38 ± 0.05% of total analyzed BM nucleated cells on average. Cells from region R2 are subsequently sorted according to the expression of CD45 antigen (marker of hematopoietic cells) as Sca-1 ⁺ /Lin ⁻ /CD45 ⁻ (region R3) and Sca-1 ⁺ /Lin ⁻ /CD45 ⁺ (region R4) subpopulations ( Fig. 12.3 C) that contain VSELs and hematopoietic stem cells (HSCs), respectively. VSELs comprise ~ 0.03% and HSCs ~ 0.35% of total BM nucleated cells ( Fig. 12.3 C). It was found that 95% of Sca-1 ⁺ /Lin ⁻ /CD45 ⁻ (VSELs) are located within the 2–6 μm size range, while 86% of Sca-1 ⁺ /Lin ⁻ /CD45 ⁺ (HSCs) are found in the 6–10 μm size range . Thus, using flow cytometry and the size marker beads, it was confirmed that the majority of Sca-1 ⁺ /Lin ⁻ /CD45 ⁻ cells isolated from adult BM are unusually small (< 6 μm). In general, VSELs are larger than peripheral blood platelets and smaller than erythrocytes. Direct transmission electronic microscopy (TEM) analysis revealed that these cells display several features typical for ESCs such as small size, a large nucleus surrounded by a narrow rim of cytoplasm and open-type chromatin (euchromatin) .

Despite their small size, VSELs possess diploid DNA. They do not express major histocompatibility complex-I (MHC-I) and HLA-DR antigens and CD90 ⁻ CD105 ⁻ CD29 ⁻ . Thus, on one hand, VSELs do not express histocompatibility antigens (MHC-I and HLA-DR) similarly to ESCs and, on the other, they do not express typical markers for mesenchymal stem cells (MSCs) (CD90 ⁻ CD105 ⁻ CD29 ⁻ ) ( Table 12.1 ). This further supports their unique phenotype.

Recently, evidence has also mounted to suggest that similar cells are also present in human umbilical cord blood (UCB) and BM, particularly in young patients ( Table 12.1 ). Accordingly, using a novel two-step isolation procedure, i.e. removal of erythrocytes by hypotonic lysis combined with multiparameter sorting, a population of human cells was isolated that is similar to VSELs previously described in murine BM . These CB-isolated (CB) VSELs are very small (4–6 μm), are highly enriched in a population of CXCR4 ⁺ AC133 ⁺ CD34 ⁺ lin ⁻ CD45 ⁻ UCB mononuclear cells (MNCs), possess large nuclei containing unorganized euchromatin, and express nuclear embryonic transcription factors Oct-4 and Nanog as well as surface SSEA-4 protein. Further studies are needed to determine whether human UCB-VSELs are endowed with PSC properties similarly to their murine BM-derived counterparts. At this point, it was possible to differentiate them in cultures over OP9 cells into early HSCs . The presence of very small cells with ESC markers (Oct-4 and SSEA-4) that are able to grow neurospheres was recently confirmed in UCB by another group of investigators .

Regeneration Potential of Very Small Embryonic-like Stem Cells

The data indicate that if plated over a C2C12 murine myoblastic cell line feeder layer, ~ 5–10% of purified VSELs are able to form spheres that resemble embryoid bodies that could be grown from established ESCs . Cells from these VSEL-derived spheres (VSEL-DSs) are composed of immature cells with large nuclei containing euchromatin and are CXCR4 ⁺ SSEA-1 ⁺ Oct-4 ⁺ , just like purified VSELs. Similar spheres were also formed by VSELs isolated from murine fetal liver, spleen and thymus. The formation of VSEL-DSs was associated with a young age in mice and no VSEL-DSs were observed in cells isolated from older mice (> 2 years) . This age-dependent content of VSELs in BM may explain why the regeneration process is more efficient in younger individuals. There are also differences in the content of these cells among BM MNCs between long- and short-lived mouse strains. The concentration of these cells is much higher in the BM of long-lived (e.g. C57Bl6) than in short-lived (DBA/2J) mice . It would be interesting to identify the genes responsible for tissue distribution and expansion of these cells, as they could be involved in controlling the life span of mammals.

Because VSELs express several markers of PGCs (fetal-type alkaline phosphatase, Oct-4, SSEA-1, CXCR4, Mvh, Stella, Fragilis, Nobox, Hdac6), they could be closely related to a population of epiblast/germ-line-derived PGCs . VSELs are also highly mobile and respond robustly to an SDF-1 gradient, adhere to fibronectin and fibrinogen, and may interact with BM-derived stromal fibroblasts . Confocal microscopy and time-lapse studies revealed that these cells attach rapidly to, migrate beneath or reside in invaginations formed by cell membranes of marrow-derived fibroblasts (emperipolesis). Because fibroblasts secrete SDF-1 and other chemoattractants, they may create a homing environment for small CXCR4 ⁺ VSELs. This robust interaction of VSELs with BM-derived fibroblasts has an important implication, namely that isolated BM stromal cells may be contaminated by these tiny cells from the beginning.

It is anticipated that VSELs could become an important source of PSCs for regeneration. Thus, researchers working with animal models must determine whether these cells could be efficiently employed in the clinic or whether they are merely developmental remnants found in the BM that cannot be harnessed effectively for regeneration. Preliminary data indicate that they play a role in regeneration of myocardium in experimental model of murine myocardial infarction if injected directly into the infarcted area. Furthermore, VSEL isolated freshly from the BM do not possess immediate hematopoietic activity (they neither grow hematopoietic colonies nor radioprotect lethally irradiated recipients); however, if plated over a supportive OP9 cell line, these CD45 ⁻ VSELs give rise to colonies of CD45 ⁺ CD41 ⁺ Gr-1 ⁻ Ter119 ⁻ cells whose phenotype resembles that of the earliest hematopoietic cells that are derived in vitro from established embryonic cell lines . This hematopoietic differentiation of VSELs was accompanied by upregulation of mRNA for several genes regulating hematopoiesis (e.g. PU-1 , c-myb , LMO2 , Ikaros ). More importantly, the CD45 ⁺ CD41 ⁻ Gr-1 ⁻ Ter119 ⁻ cells expanded from VSEL isolated from GFP ⁺ mice, when transplanted into wild-type animals, protected them from lethal irradiation and differentiated in vivo into all major hematopoietic lineages (e.g. Gr-1 ⁺ , B220 ⁺ and CD3 ⁺ cells) . Thus, it is proposed that VSELs are a population of BM-residing PSCs that may give rise to long-term engrafting HSCs.