Kasia Mierzejewska

Ceramide-1-Phosphate Regulates Migration of Multipotent Stromal Cells and Endothelial Progenitor Cells—Implications for Tissue Regeneration†‡§

Ceramide‐1‐phosphate (C1P) is a bioactive lipid that, in contrast to ceramide, is an antiapoptotic molecule released from cells that are damaged and “leaky.” As reported recently, C1P promotes migration of hematopoietic cells. In this article, we tested the hypothesis that C1P released upon tissue damage may play an underappreciated role in chemoattraction of various types of stem cells and endothelial cells involved in tissue/organ regeneration. We show for the first time that C1P is upregulated in damaged tissues and chemoattracts bone marrow (BM)‐derived multipotent stromal cells, endothelial progenitor cells, and very small embryonic‐like stem cells. Furthermore, compared to other bioactive lipids, C1P more potently chemoattracted human umbilical vein endothelial cells and stimulated tube formation by these cells. C1P also promoted in vivo vascularization of Matrigel implants and stimulated secretion of stromal cell‐derived factor‐1 from BM‐derived fibroblasts. Thus, our data demonstrate, for the first time, that C1P is a potent bioactive lipid released from damaged cells that potentially plays an important and novel role in recruitment of stem/progenitor cells to damaged organs and may promote their vascularization. STEM CELLS2013;31:500–510

Paracrine proangiopoietic effects of human umbilical cord blood-derived purified CD133+ cells--implications for stem cell therapies in regenerative medicine.

CD133+ cells purified from hematopoietic tissues are enriched mostly for hematopoietic stem/progenitor cells, but also contain some endothelial progenitor cells and very small embryonic-like stem cells. CD133+ cells, which are akin to CD34+ cells, are a potential source of stem cells in regenerative medicine. However, the lack of convincing donor-derived chimerism in the damaged organs of patients treated with these cells suggests that the improvement in function involves mechanisms other than a direct contribution to the damaged tissues. We hypothesized that CD133+ cells secrete several paracrine factors that play a major role in the positive effects observed after treatment and tested supernatants derived from these cells for the presence of such factors. We observed that CD133+ cells and CD133+ cell-derived microvesicles (MVs) express mRNAs for several antiapoptotic and proangiopoietic factors, including kit ligand, insulin growth factor-1, vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8. These factors were also detected in a CD133+ cell-derived conditioned medium (CM). More important, the CD133+ cell-derived CM and MVs chemoattracted endothelial cells and display proangiopoietic activity both in vitro and in vivo assays. This observation should be taken into consideration when evaluating clinical outcomes from purified CD133+ cell therapies in regenerative medicine.

The Proper Criteria for Identification and Sorting of Very Small Embryonic-Like Stem Cells, and Some Nomenclature Issues

Evidence has accumulated that both murine and human adult tissues contain early-development stem cells with a broader differentiation potential than other adult monopotent stem cells. These cells, being pluripotent or multipotent, exist at different levels of specification and most likely represent overlapping populations of cells that, depending on the isolation strategy, ex vivo expansion protocol, and markers employed for their identification, have been given different names. In this review, we will discuss a population of very small embryonic-like stem cells (VSELs) in the context of other stem cells that express pluripotent/multipotent markers isolated from adult tissues as well as review the most current, validated working criteria on how to properly identify and isolate these very rare cells. VSELs have been successfully purified in several laboratories; however, a few have failed to isolate them, which has raised some unnecessary controversy in the field. Therefore, in this short review, we will address the most important reasons that some investigators have experienced problems in isolating these very rare cells and discuss some still unresolved challenges which should be overcome before these cells can be widely employed in the clinic.

Very small embryonic-like stem-cell optimization of isolation protocols: an update of molecular signatures and a review of current in vivo applications.

As the theory of stem cell plasticity was first proposed, we have explored an alternative hypothesis for this phenomenon: namely that adult bone marrow (BM) and umbilical cord blood (UCB) contain more developmentally primitive cells than hematopoietic stem cells (HSCs). In support of this notion, using multiparameter sorting we were able to isolate small Sca1+Lin−CD45− cells and CD133+Lin−CD45− cells from murine BM and human UCB, respectively, which were further enriched for the detection of various early developmental markers such as the SSEA antigen on the surface and the Oct4 and Nanog transcription factors in the nucleus. Similar populations of cells have been found in various organs by our team and others, including the heart, brain and gonads. Owing to their primitive cellular features, such as the high nuclear/cytoplasm ratio and the presence of euchromatin, they are called very small embryonic-like stem cells (VSELs). In the appropriate in vivo models, VSELs differentiate into long-term repopulating HSCs, mesenchymal stem cells (MSCs), lung epithelial cells, cardiomyocytes and gametes. In this review, we discuss the most recent data from our laboratory and other groups regarding the optimal isolation procedures and describe the updated molecular characteristics of VSELs.

Sphingosine-1-phosphate-Mediated Mobilization of Hematopoietic Stem/Progenitor Cells during Intravascular Hemolysis Requires Attenuation of SDF-1-CXCR4 Retention Signaling in Bone Marrow

Sphingosine-1-phosphate (S1P) is a crucial chemotactic factor in peripheral blood (PB) involved in the mobilization process and egress of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM). Since S1P is present at high levels in erythrocytes, one might assume that, by increasing the plasma S1P level, the hemolysis of red blood cells would induce mobilization of HSPCs. To test this assumption, we induced hemolysis in mice by employing phenylhydrazine (PHZ). We observed that doubling the S1P level in PB from damaged erythrocytes induced only a marginally increased level of mobilization. However, if mice were exposed to PHZ together with the CXCR4 blocking agent, AMD3100, a robust synergistic increase in the number of mobilized HSPCs occurred. We conclude that hemolysis, even if it significantly elevates the S1P level in PB, also requires attenuation of the CXCR4-SDF-1 axis-mediated retention in BM niches for HSPC mobilization to occur. Our data also further confirm that S1P is a major chemottractant present in plasma and chemoattracts HSPCs into PB under steady-state conditions. However, to egress from BM, HSPCs first have to be released from BM niches by blocking the SDF-1-CXCR4 retention signal.

CD133 Expression Strongly Correlates with the Phenotype of Very Small Embryonic-/Epiblast-Like Stem Cells.

CD133 antigen (prominin-1) is a useful cell surface marker of very small embryonic-like stem cells (VSELs). Antibodies against it, conjugated to paramagnetic beads or fluorochromes, are thus powerful biological tools for their isolation from human umbilical cord blood, mobilized peripheral blood, and bone marrow. VSELs are described with the following characteristics: (1) are slightly smaller than red blood cells; (2) display a distinct morphology, typified by a high nuclear/cytoplasmic ratio and an unorganized euchromatin; (3) become mobilized during stress situations into peripheral blood; (4) are enriched in the CD133(+)Lin(-)CD45(-) cell fraction in humans; and (5) express markers of pluripotent stem cells (e.g., Oct-4, Nanog, and stage-specific embryonic antigen-4). The most recent in vivo data from our and other laboratories demonstrated that human VSELs exhibit some characteristics of long-term repopulating hematopoietic stem cells and are at the top of the hierarchy in the mesenchymal lineage. However, still more labor is needed to characterize better at a molecular level these rare cells.

Further evidence that paroxysmal nocturnal haemoglobinuria is a disorder of defective cell membrane lipid rafts

The glycolipid glycosylphosphatidylinositol anchor (GPI‐A) plays an important role in lipid raft formation, which is required for proper expression on the cell surface of two inhibitors of the complement cascade, CD55 and CD59. The absence of these markers from the surface of blood cells, including erythrocytes, makes the cells susceptible to complement lysis, as seen in patients suffering from paroxysmal nocturnal haemoglobinuria (PNH). However, the explanation for why PNH‐affected hematopoietic stem/progenitor cells (HSPCs) expand over time in BM is still unclear. Here, we propose an explanation for this phenomenon and provide evidence that a defect in lipid raft formation in HSPCs leads to defective CXCR4‐ and VLA‐4‐mediated retention of these cells in BM. In support of this possibility, BM‐isolated CD34+ cells from PNH patients show a defect in the incorporation of CXCR4 and VLA‐4 into membrane lipid rafts, respond weakly to SDF‐1 stimulation, and show defective adhesion to fibronectin. Similar data were obtained with the GPI‐A− Jurkat cell line. Moreover, we also report that chimeric mice transplanted with CD55−/− CD59−/− BM cells but with proper GPI‐A expression do not expand over time in transplanted hosts. On the basis of these findings, we propose that a defect in lipid raft formation in PNH‐mutated HSPCs makes these cells more mobile, so that they expand and out‐compete normal HSPCs from their BM niches over time.

Microvesicles and Their Emerging Role in Cellular Therapies for Organ and Tissue Regeneration

Microvesicles (MVs) are small, spherical membrane fragments shed from the cell surface or secreted from the endosomal compartment. MVs released from cells employed in regenerative medicine to rescue damaged tissues seem to play an important and underappreciated role in improving the function of damaged organs. A growing body of evidence suggests that MVs secreted from hematopoietic stem progenitor cells (HSPCs), multipotent stroma cells (MSCs), or cardiac stem cells (CSCs) employed in various treatment strategies in regenerative medicine may: (1) inhibit apoptosis of cells residing in the damaged tissues, (2) stimulate proliferation of cells that survive organ injury, and (3) stimulate vascularization of affected tissues. These proregenerative effects mediated by MVs are explained by the fact that these small, spherical membrane fragments: (1) are enriched in bioactive lipids (e.g., sphingosine-1-phosphate [S1P]), (2) may express antiapoptoic and prostimulatory growth factors or cytokines (e.g., vascular endothelial growth factor [VEGF], stem cell factor [SCF], or stromal derived factor-1 [SDF-1]) on their surface, and (3) may deliver mRNA, regulatory miRNA, and proteins to the damaged tissues that improve overall cell function. Based on these observations, the potential use of MVs, instead of whole cells, has become an exciting new concept in regenerative medicine.