Wan Wu

Evidence That Very Small Embryonic‐Like Stem Cells Are Mobilized into Peripheral Blood

Recently, we identified in murine adult tissues, including bone marrow, a population of very small embryonic‐like (VSEL) stem cells. Here, we provide further evidence that under steady‐state conditions these cells circulate at very low levels in peripheral blood (PB) (∼100–200 cells/ml) and could be additionally mobilized during pharmacological granulocyte‐colony‐stimulating factor‐induced or stress‐related mobilization, as demonstrated in a model of toxic liver or skeletal muscle damage induced by injection of carbon tetrachloride or cardiotoxin, respectively. The number of circulating VSEL stem cells under steady‐state conditions in PB of 2‐month‐old animals was five times higher than that in 1‐year‐old mice. In conclusion, this study supports a hypothesis that VSEL stem cells are a mobile pool of primitive stem cells that could be released from the stem cell niches into PB. Further studies are needed, however, to see whether the level of these cells circulating in PB could become a prognostic indicator to assess the regenerative potential of an adult organism and/or clinical outcome from an injury.

Very small embryonic-like stem cells are present in adult murine organs: ImageStream-based morphological analysis and distribution studies†

Recently, we purified a population of CXCR4+/Oct‐4+/SSEA‐1+/Sca‐1+/Lin−/CD45− very small embryonic‐like stem cells (VSELs) from adult murine bone marrow (BM). After using flow cytometry, ImageStream analysis, confocal microscopy, and real time RT‐PCR, we report that similar cells could be also identified and isolated from several organs in adult mice. The highest total numbers of Oct‐4+ VSELs were found in the brain, kidneys, muscles, pancreas, and BM. These observations support our hypothesis that a population of very primitive cells expressing germ line/epiblast markers (Oct‐4, SSEA‐1) is deposited early during embryogenesis in various organs and survives into adulthood. Further studies are needed to determine whether these cells, after being isolated from various adult human organs similarly to their murine BM‐derived counterparts, are endowed with pluripotent stem cell properties.

Impaired mobilization of hematopoietic stem/progenitor cells in C5-deficient mice supports the pivotal involvement of innate immunity in this process and reveals novel promobilization effects of granulocytes

We reported that complement cascade (CC) becomes activated in bone marrow (BM) during granulocyte colony-stimulating factor (G-CSF) mobilization of hematopoietic stem/progenitor cells (HSPCs) and showed that, although third CC component (C3)-deficient mice are easy mobilizers, fifth CC component (C5)-deficient mice mobilize very poorly. To explain this, we postulated that activation/cleavage of CC releases C3a and C5a anaphylatoxins that differently regulate mobilization. Accordingly, C3a, by enhancing responsiveness of HSPCs to decreasing concentrations of stromal-derived growth factor-1 (SDF-1) in BM, prevents mobilization and promotes their BM retention. Therefore, in this study, we focused on the mobilization-enhancing role of C5a. We found that C5a receptor (C5aR) is not expressed on the surface of HSPCs, and that C5a-mediated promobilization effects are mediated by stimulation of granulocytes. Overall, our data support the following model. First C5aR+ granulocytes are chemoattracted by plasma C5 cleavage fragments, being the first wave of cells leaving BM. This facilitates a subsequent egress of HSPCs. In the next step, after leaving BM, granulocytes undergo degranulation in response to plasma C5a and secrete some cationic peptides (cathelicidin, β-defensin) that, as shown here for the first time, highly enhance the responsiveness of HSPCs to plasma SDF-1 gradient. In conclusion, our data reveal the underappreciated central role of innate immunity in mobilization, in which C5 cleavage fragments through granulocytes orchestrate this process.

Conditioning for hematopoietic transplantation activates the complement cascade and induces a proteolytic environment in bone marrow: a novel role for bioactive lipids and soluble C5b-C9 as homing factors.

We have observed that conditioning for hematopoietic transplantation by lethal irradiation induces a proteolytic microenvironment in the bone marrow (BM) that activates the complement cascade (CC). As a result, BM is enriched for proteolytic enzymes and the soluble form of the terminal product of CC activation, the membrane attack complex C5b-C9 (MAC). At the same time, proteolytic enzymes induced in irradiated BM impair the chemotactic activity of α-chemokine stromal-derived factor-1 (SDF-1). As SDF-1 is considered a crucial BM chemoattractant for transplanted hematopoietic stem/progenitor cells (HSPCs), we sought to determine whether other factors that are resistant to proteolytic enzymes have a role in this process, focusing on proteolysis-resistant bioactive lipids. We found that the concentrations of sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) increase in the BM after conditioning for transplantation and that both S1P and, as we show here for the first time, C1P are potent chemoattractants for HSPCs. Next, we observed that C5-deficient mice that do not generate MAC show impaired engraftment of HSPCs. In support of a role for MAC in homing and engraftment, we found that soluble MAC enhances in a CR3 (CD11b/CD18)-dependent manner the adhesion of HSPCs to BM stromal cells and increases the secretion of SDF-1 by BM stroma. We conclude that an increase in BM levels of proteolytic enzyme-resistant S1P and C1P and activation of CC, which leads to the generation of MAC, has an important and previously underappreciated role in the homing of transplanted HSPCs.

Molecular signature of adult bone marrow-purified very small embryonic-like stem cells supports their developmental epiblast/germ line origin

We postulated that Oct4+SSEA-1+Sca-1+Lin−CD45− very small embryonic-like stem cells (VSELs) isolated from adult bone marrow (BM) could be a reserve population for tissue-committed stem cells. The aim of this study was to elucidate the developmental origin of these cells. We report that during embryogenesis, VSELs are enriched in embryonic day (E)12.5 murine fetal livers (FLs) and subsequently follow the developmental route of hematopoietic stem cells (H)SCs to colonize BM. Molecular analysis of purified VSELs revealed that both FL-derived VSELs and their adult BM-derived counterparts express: (i) several epiblast/primordial germ cell (PGC) markers; (ii) migrating PGC-like epigenetic reprogramming profiles of Oct4, Nanog and Stella loci; as well as (iii) a unique pattern of genomic imprinting. Thus, these data suggest that VSELs may originate from epiblast/migrating PGC-like cells and, in spite of the expression of pluripotent stem cell markers, changes in the epigenetic signature of imprinted genes keep these cells quiescent in adult tissues and prevent them from teratoma formation.

The bone marrow-expressed antimicrobial cationic peptide LL-37 enhances the responsiveness of hematopoietic stem progenitor cells to an SDF-1 gradient and accelerates their engraftment after transplantation

We report that the bone marrow (BM) stroma-released LL-37, a member of the cathelicidin family of antimicrobial peptides, primes/increases the responsiveness of murine and human hematopoietic stem/progenitor cells (HSPCs) to an α-chemokine stromal-derived factor-1 (SDF-1) gradient. Accordingly, LL-37 is upregulated in irradiated BM cells and enhances the chemotactic responsiveness of hematopoietic progenitors from all lineages to a low physiological SDF-1 gradient as well as increasing their (i) adhesiveness, (ii) SDF-1-mediated actin polymerization and (iii) MAPKp42/44 phosphorylation. Mice transplanted with BM cells ex vivo primed by LL-37 showed accelerated recovery of platelet and neutrophil counts by ∼3–5 days compared with mice transplanted with unprimed control cells. These priming effects were not mediated by LL-37 binding to its receptor and depended instead on the incorporation of the CXCR4 receptor into membrane lipid rafts. We propose that LL-37, which has primarily antimicrobial functions and is harmless to mammalian cells, could be clinically applied to accelerate engraftment as an ex vivo priming agent for transplanted human HSPCs. This novel approach would be particularly important in cord blood transplantations, where the number of HSCs available is usually limited.

Global Gene Expression Analysis of Very Small Embryonic-Like Stem Cells Reveals that the Ezh2-Dependent Bivalent Domain Mechanism Contributes to Their Pluripotent State

Recently, we identified a population of Oct4(+)Sca-1(+)Lin(-)CD45(-) very small embryonic-like stem cells (VSELs) in murine and human adult tissues. VSELs can differentiate in vitro into cells from all 3 germ layers and in vivo tissue-committed stem cells. Open chromatin structure of core pluripotency transcription factors (TFs) supports the pluripotent state of VSELs. However, it has been difficult to determine how primitive VSELs maintain pluripotency, owing to their limited number in adult tissues. Here, we demonstrate by genome-wide gene-expression analysis with a small number of highly purified murine bone marrow-derived VSELs that Oct4(+) VSELs (i) express a similar, yet nonidentical, transcriptome as embryonic stem cells (ESCs), (ii) highly express cell cycle checkpoint genes, (iii) express at a low level genes involved in protein turnover and mitogenic pathways, and (iv) highly express enhancer of zeste drosophila homolog 2 (Ezh2), a polycomb group protein. Furthermore, as a result of high expression of Ezh2, VSELs, like ESCs, exhibit bivalently modified nucleosomes (trimethylated H3K27 and H3K4) at promoters of important homeodomain-containing developmental TFs, thus preventing premature activation of the lineage-committing factors. Notably, spontaneous or RNA interference-enforced downregulation of Ezh2 during VSEL differentiation removes the bivalent domain (BD) structure, which leads to de-repression of several BD-regulated genes. Therefore, we suggest that Oct4(+) VSELs, like other pluripotent stem cells, maintain their pluripotent state through an Ezh2-dependent BD-mediated epigenetic mechanism. Furthermore, our global survey of VSEL gene expression signature would not only advance our understanding of biological process for their pluripotency, differentiation, and quiescence but should also help to develop better protocols for ex vivo expansion of VSELs.

Defective engraftment of C3aR-/- hematopoietic stem progenitor cells shows a novel role of the C3a-C3aR axis in bone marrow homing

We reported that complement (C) becomes activated and cleaved in bone marrow during preconditioning for hematopoietic transplantation and the third C component (C3) cleavage fragments, C3a and desArgC3a, increase responsiveness of hematopoietic stem/progenitor cells (HSPCs) to stromal-derived factor-1 (SDF-1). We also showed that this homing-promoting effect is not C3a receptor (C3aR) dependent. Herein, we report our new observation that transplantation of C3aR−/− HSPCs into lethally irradiated recipients results in: (1) ∼5–7 day delay in recovery of platelets and leukocytes; (2) decrease in formation of day 12 colony-forming units-spleen; and (3) decrease in the number of donor-derived CFU-granulocyte-macrophage progenitors detectable in the bone marrow cavities at day 16 after transplantation. In agreement with the murine data, blockage of C3aR on human umbilical cord blood CD34+ cells by C3aR antagonist SB290157 impairs their engraftment in non-obese diabetic/severe combined immunodeficient mice. However, HSPCs from C3aR−/− mice stimulated by C3a still better responded to SDF-1 gradient, after exposure to C3a, they secrete less matrix metalloprotease-9 and show impaired adhesion to stroma cells. We conclude that C3a, in addition to enhancing responsiveness of HSPCs to SDF-1 gradient in a C3aR independent manner, may also directly modulate HSPC homing by augmenting C3aR-mediated secretion of matrix metalloprotease-9 and cell adhesion.

Bone marrow-derived very small embryonic-like stem cells: Their developmental origin and biological significance

Data from our and other laboratories provide evidence that bone marrow (BM) contains a population of stem cells that expresses early developmental markers such as (1) stage‐specific embryonic antigen (SSEA) and (2) transcription factors Oct‐4 and Nanog. These are the markers characteristic for embryonic stem cells, epiblast stem cells, and primordial germ cells (PGC). The presence of these stem cells in adult BM supports the concept that this organ contains some population of pluripotent stem cells that is deposited in embryogenesis during early gastrulation. We hypothesize that these cells could be direct descendants of the germ lineage that, to pass genes on to the next generations, has to create soma and, thus, becomes a “mother lineage” for all somatic cell lineages present in the adult body. Germ potential is established after conception in totipotent zygotes and retained in blastomeres of morula, cells from the inner cell mass of blastocyst, epiblast, and population of PGC. We will present a concept that SSEA+ Oct‐4+ Nanog+ cells identified in BM could be descendants of epiblast cells as well as some rare migrating astray PGC. Developmental Dynamics 236:3309–3320, 2007.

Adult marrow-derived very small embryonic-like stem cells and tissue engineering

A population of CXCR4+ lin- CD45- cells that express SSEA, Oct-4 and Nanog has been identified in adult bone marrow. These cells are very small and display several features typical for primary embryonic stem cells such as: i) a large nuclei surrounded by a narrow rim of cytoplasm; ii) open-type chromatin (euchromatin); and iii) high telomerase activity. These cells were named very small embryonic-like stem cells (VSEL-SC). The authors hypothesized that they are direct descendants of the germ lineage. Germ lineage, in order to pass genes on to the next generation, has to create soma and thus becomes a ‘mother lineage’ for all somatic cell lineages present in the adult body. Germ potential is established after conception in a totipotent zygote and retained subsequently during development in blastomers of morula, cells form the inner cell mass of blastocyst, epiblast and population of primordial germ cells. The authors envision that VSEL-SC are epiblast-derived pluripotent stem cells and could potentially become a less-controversial source of stem cells for regeneration.