Janina Ratajczak

Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication.

Normal and malignant cells shed from their surface membranes as well as secrete from the endosomal membrane compartment circular membrane fragments called microvesicles (MV). MV that are released from viable cells are usually smaller in size compared to the apoptotic bodies derived from damaged cells and unlike them do not contain fragmented DNA. Growing experimental evidence indicates that MV are an underappreciated component of the cell environment and play an important pleiotropic role in many biological processes. Generally, MV are enriched in various bioactive molecules and may (i) directly stimulate cells as a kind of ‘signaling complex’, (ii) transfer membrane receptors, proteins, mRNA and organelles (e.g., mitochondria) between cells and finally (iii) deliver infectious agents into cells (e.g., human immuno deficiency virus, prions). In this review, we discuss the pleiotropic effects of MV that are important for communication between cells, as well as the role of MV in carcinogenesis, coagulation, immune responses and modulation of susceptibility/infectability of cells to retroviruses or prions.

Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery

Membrane-derived vesicles (MV) are released from the surface of activated eucaryotic cells and exert pleiotropic effects on surrounding cells. Since the maintenance of pluripotency and undifferentiated propagation of embryonic stem (ES) cells in vitro requires tight cell to cell contacts and effective intercellular signaling, we hypothesize that MV derived from ES cells (ES-MV) express stem cell-specific molecules that may also support self-renewal and expansion of adult stem cells. To address this hypothesis, we employed expansion of hematopoietic progenitor cells (HPC) as a model. We found that ES-MV (10 μg/ml) isolated from murine ES cells (ES-D3) in serum-free cultures significantly (i) enhanced survival and improved expansion of murine HPC, (ii) upregulated the expression of early pluripotent (Oct-4, Nanog and Rex-1) and early hematopoietic stem cells (Scl, HoxB4 and GATA 2) markers in these cells, and (iii) induced phosphorylation of MAPK p42/44 and serine-threonine kinase AKT. Furthermore, molecular analysis revealed that ES-MV express Wnt-3 protein and are selectively highly enriched in mRNA for several pluripotent transcription factors as compared to parental ES cells. More important, this mRNA could be delivered by ES-MV to target cells and translated into the corresponding proteins. The biological effects of ES-MV were inhibited after heat inactivation or pretreatment with RNAse, indicating a major involvement of protein and mRNA components of ES-MV in the observed phenomena. We postulate that ES-MV may efficiently expand HPC by stimulating them with ES-MV expressed ligands (e.g., Wnt-3) as well as increase their pluripotency after horizontal transfer of ES-derived mRNA.

Trafficking of normal stem cells and metastasis of cancer stem cells involve similar mechanisms: pivotal role of the SDF-1-CXCR4 axis.

The α‐chemokine stromal‐derived factor (SDF)‐1 and the G‐protein–coupled seven‐span transmembrane receptor CXCR4 axis regulates the trafficking of various cell types. In this review, we present the concept that the SDF‐1–CXCR4 axis is a master regulator of trafficking of both normal and cancer stem cells. Supporting this is growing evidence that SDF‐1 plays a pivotal role in the regulation of trafficking of normal hematopoietic stem cells (HSCs) and their homing/retention in bone marrow. Moreover, functional CXCR4 is also expressed on nonhematopoietic tissue‐committed stem/progenitor cells (TCSCs); hence, the SDF‐1–CXCR4 axis emerges as a pivotal regulator of trafficking of various types of stem cells in the body. Furthermore, because most if not all malignancies originate in the stem/progenitor cell compartment, cancer stem cells also express CXCR4 on their surface and, as a result, the SDF‐1–CXCR4 axis is also involved in directing their trafficking/metastasis to organs that highly express SDF‐1 (e.g., lymph nodes, lungs, liver, and bones). Hence, we postulate that the metastasis of cancer stem cells and trafficking of normal stem cells involve similar mechanisms, and we discuss here the common molecular mechanisms involved in these processes. Finally, the responsiveness of CXCR4+ normal and malignant stem cells to an SDF‐1 gradient may be regulated positively/primed by several small molecules related to inflammation which enhance incorporation of CXCR4 into membrane lipid rafts, or may be inhibited/blocked by small CXCR4 antagonist peptides. Consequently, strategies aimed at modulating the SDF‐1–CXCR4 axis could have important clinical applications both in regenerative medicine to deliver normal stem cells to the tissues/organs and in clinical hematology/oncology to inhibit metastasis of cancer stem cells.

A population of very small embryonic-like (VSEL) CXCR4+SSEA-1 +OCT-4+ stem cells identified in adult bone marrow

By employing multiparameter sorting, we identified in murine bone marrow (BM) a homogenous population of rare (∼0.02% of BMMNC) Sca-1+lin−CD45− cells that express by RQ-PCR and immunohistochemistry markers of pluripotent stem cells (PSC) such as SSEA-1, Oct-4, Nanog and Rex-1. The direct electronmicroscopical analysis revealed that these cells are small (∼2–4 μm), posses large nuclei surrounded by a narrow rim of cytoplasm, and contain open-type chromatin (euchromatin) that is typical for embryonic stem cells. In vitro cultures these cells are able to differentiate into all three germ-layer lineages. The number of these cells is highest in BM from young (∼1-month-old) mice and decreases with age. It is also significantly diminished in short living DBA/2J mice as compared to long living B6 animals. These cells in vitro respond strongly to SDF-1, HGF/SF and LIF and express CXCR4, c-met and LIF-R, respectively, and since they adhere to fibroblasts they may be coisolated with BM adherent cells. We hypothesize that this population of Sca-1+lin−CD45− very small embryonic-like (VSEL) stem cells is deposited early during development in BM and could be a source of pluripotent stem cells for tissue/organ regeneration.

CXCR4-SDF-1 signalling, locomotion, chemotaxis and adhesion.

Chemokines, small pro-inflammatory chemoattractant cytokines, that bind to specific G-protein-coupled seven-span transmembrane receptors present on plasma membranes of target cells are the major regulators of cell trafficking. In addition some chemokines have been reported to modulate cell survival and growth. Moreover, compelling evidence is accumulating that cancer cells may employ several mechanisms involving chemokine–chemokine receptor axes during their metastasis that also regulate the trafficking of normal cells. Of all the chemokines, stromal-derived factor-1 (SDF-1), an α-chemokine that binds to G-protein-coupled CXCR4, plays an important and unique role in the regulation of stem/progenitor cell trafficking. First, SDF-1 regulates the trafficking of CXCR4+ haemato/lymphopoietic cells, their homing/retention in major haemato/lymphopoietic organs and accumulation of CXCR4+ immune cells in tissues affected by inflammation. Second, CXCR4 plays an essential role in the trafficking of other tissue/organ specific stem/progenitor cells expressing CXCR4 on their surface, e.g., during embryo/organogenesis and tissue/organ regeneration. Third, since CXCR4 is expressed on several tumour cells, these CXCR4 positive tumour cells may metastasize to the organs that secrete/express SDF-1 (e.g., bones, lymph nodes, lung and liver). SDF-1 exerts pleiotropic effects regulating processes essential to tumour metastasis such as locomotion of malignant cells, their chemoattraction and adhesion, as well as plays an important role in tumour vascularization. This implies that new therapeutic strategies aimed at blocking the SDF-1–CXCR4 axis could have important applications in the clinic by modulating the trafficking of haemato/lymphopoietic cells and inhibiting the metastatic behaviour of tumour cells as well. In this review, we focus on a role of the SDF-1–CXCR4 axis in regulating the metastatic behaviour of tumour cells and discuss the molecular mechanisms that are essential to this process.

Migration of Bone Marrow and Cord Blood Mesenchymal Stem Cells In Vitro Is Regulated by Stromal‐Derived Factor‐1‐CXCR4 and Hepatocyte Growth Factor‐c‐met Axes and Involves Matrix Metalloproteinases

Human mesenchymal stem cells (MSCs) are increasingly being considered in cell‐based therapeutic strategies for regeneration of various organs/tissues. However, the signals required for their homing and recruitment to injured sites are not yet fully understood. Because stromal‐derived factor (SDF)‐1 and hepatocyte growth factor (HGF) become up‐regulated during tissue/organ damage, in this study we examined whether these factors chemoattract ex vivo‐expanded MSCs derived from bone marrow (BM) and umbilical cord blood (CB). Specifically, we investigated the expression by MSCs of CXCR4 and c‐met, the cognate receptors of SDF‐1 and HGF, and their functionality after early and late passages of MSCs. We also determined whether MSCs express matrix metalloproteinases (MMPs), including membrane type 1 (MT1)‐MMP, matrix‐degrading enzymes that facilitate the trafficking of hematopoietic stem cells. We maintained expanded BM‐ or CB‐derived MSCs for up to 15–18 passages with monitoring of the expression of 1) various tissue markers (cardiac and skeletal muscle, neural, liver, and endothelial cells), 2) functional CXCR4 and c‐met, and 3) MMPs. We found that for up to 15–18 passages, both BM‐ and CB‐derived MSCs 1) express mRNA for cardiac, muscle, neural, and liver markers, as well as the vascular endothelial (VE) marker VE‐cadherin; 2) express CXCR4 and c‐met receptors and are strongly attracted by SDF‐1 and HGF gradients; 3) express MMP‐2 and MT1‐MMP transcripts and proteins; and 4) are chemo‐invasive across the reconstituted basement membrane Matrigel. These in vitro results suggest that the SDF‐1‐CXCR4 and HGF‐c‐met axes, along with MMPs, may be involved in recruitment of expanded MSCs to damaged tissues.

Microvesicles derived from activated platelets induce metastasis and angiogenesis in lung cancer.

The role of platelets in tumor progression and metastasis has been recognized but the mechanism of their action remains unclear. Five human lung cancer cell lines (A549, CRL 2066, CRL 2062, HTB 183, HTB 177) and a murine Lewis lung carcinoma (LCC) cell line (for an in vivo model of metastasis) were used to investigate how platelet‐derived microvesicles (PMV), which are circular fragments shed from the surface membranes of activated platelets, and exosomes released from platelet α‐granules, could contribute to metastatic spread. We found that PMV transferred the platelet‐derived integrin CD41 to most of the lung cancer cell lines tested and stimulated the phosphorylation of mitogen‐activated protein kinase p42/44 and serine/threonine kinase as well as the expression of membrane type 1‐matrix metalloproteinase (MT1‐MMP). PMV chemoattracted 4 of the 5 cell lines, with the highly metastatic A549 cells exhibiting the strongest response. In A549 cells, PMV were shown to stimulate proliferation, upregulate cyclin D2 expression and increase trans‐Matrigel chemoinvasion. Furthermore, in these cells, PMV stimulated mRNA expression for angiogenic factors such as MMP‐9, vascular endothelial growth factor, interleukin‐8 and hepatocyte growth factor, as well as adhesion to fibrinogen and human umbilical vein endothelial cells. Intravenous injection of murine PMV‐covered LLC cells into syngeneic mice resulted in significantly more metastatic foci in their lungs and LLC cells in bone marrow than in control animals injected with LCC cells not covered with PMV. Based on these findings, we suggest that PMV play an important role in tumor progression/metastasis and angiogenesis in lung cancer.

Stem cell plasticity revisited: CXCR4-positive cells expressing mRNA for early muscle, liver and neural cells ‘hide out’ in the bone marrow

It has been suggested that bone marrow (BM)-derived hematopoietic stem cells transdifferentiate into tissue-specific stem cells (the so-called phenomenon of stem cell plasticity), but the possibility of committed tissue-specific stem cells pre-existing in BM has not been given sufficient consideration. We hypothesized that (i) tissue-committed stem cells circulate at a low level in the peripheral blood (PB) under normal steady-state conditions, maintaining a pool of stem cells in peripheral tissues, and their levels increase in PB during stress/tissue injury, and (ii) they could be chemoattracted to the BM where they find a supportive environment and that the SDF-1–CXCR4 axis plays a prominent role in the homing/retention of these cells to BM niches. We performed all experiments using freshly isolated cells to exclude the potential for ‘transdifferentiation’ of hematopoietic stem or mesenchymal cells associated with in vitro culture systems. We detected mRNA for various early markers for muscle (Myf-5, Myo-D), neural (GFAP, nestin) and liver (CK19, fetoprotein) cells in circulating (adherent cell-depleted) PB mononuclear cells (MNC) and increased levels of expression of these markers in PB after mobilization by G-CSF (as measured using real-time RT-PCR). Furthermore, SDF-1 chemotaxis combined with real-time RT-PCR analysis revealed that (i) these early tissue-specific cells reside in normal murine BM, (ii) express CXCR4 on their surface and (iii) can be enriched (up to 60 ×) after chemotaxis to an SDF-1 gradient. These cells were also highly enriched within purified populations of murine Sca-1+ BM MNC as well as of human CD34+-, AC133+- and CXCR4-positive cells. We also found that the expression of mRNA for SDF-1 is upregulated in damaged heart, kidney and liver. Hence our data provide a new perspective on BM not only as a home for hematopoietic stem cells but also a ‘hideout’ for already differentiated CXCR4-positive tissue-committed stem/progenitor cells that follow an SDF-1 gradient, could be mobilized into PB, and subsequently take part in organ/tissue regeneration.

The pleiotropic effects of the SDF-1-CXCR4 axis in organogenesis, regeneration and tumorigenesis.

Proper response of normal stem cells (NSC) to motomorphogens and chemoattractants plays a pivotal role in organ development and renewal/regeneration of damaged tissues. Similar chemoattractants may also regulate metastasis of cancer stem cells (CSC). Growing experimental evidence indicates that both NSC and CSC express G-protein-coupled seven-transmembrane span receptor CXCR4 and respond to its specific ligand α-chemokine stromal derived factor-1 (SDF-1), which is expressed by stroma cells from different tissues. In addition, a population of very small embryonic-like (VSEL) stem cells that express CXCR4 and respond robustly to an SDF-1 gradient was recently identified in adult tissues. VSELs express several markers of embryonic and primordial germ cells. It is proposed that these cells are deposited early in the development as a dormant pool of embryonic/pluripotent NSC. Expression of both CXCR4 and SDF-1 is upregulated in response to tissue hypoxia and damage signal attracting circulating NSC and CSC. Thus, pharmacological modulation of the SDF-1–CXCR4 axis may lead to the development of new therapeutic strategies to enhance mobilization of CXCR4+ NSC and their homing to damaged organs as well as inhibition of the metastasis of CXCR4+ cancer cells.

Cells Expressing Early Cardiac Markers Reside in the Bone Marrow and Are Mobilized Into the Peripheral Blood After Myocardial Infarction

The concept that bone marrow (BM)–derived cells participate in cardiac regeneration remains highly controversial and the identity of the specific cell type(s) involved remains unknown. In this study, we report that the postnatal BM contains a mobile pool of cells that express early cardiac lineage markers (Nkx2.5/Csx, GATA-4, and MEF2C). These cells are present in significant amounts in BM harvested from young mice but their abundance decreases with age; in addition, the responsiveness of these cells to gradients of motomorphogens SDF-1, HGF, and LIF changes with age. FACS analysis, combined with analysis of early cardiac markers at the mRNA and protein levels, revealed that cells expressing these markers reside in the nonadherent, nonhematopoietic CXCR4+/Sca-1+/lin−/CD45− mononuclear cell (MNC) fraction in mice and in the CXCR4+/CD34+/AC133+/CD45− BMMNC fraction in humans. These cells are mobilized into the peripheral blood after myocardial infarction and chemoattracted to the infarcted myocardium in an SDF-1-CXCR4–, HGF-c-Met–, and LIF-LIF-R–dependent manner. To our knowledge, this is the first demonstration that the postnatal BM harbors a nonhematopoietic population of cells that express markers for cardiac differentiation. We propose that these potential cardiac progenitors may account for the myocardial regenerative effects of BM. The present findings provide a novel paradigm that could reconcile current controversies and a rationale for investigating the use of BM-derived cardiac progenitors for myocardial regeneration.