Bogdan Machalinski

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.

Morphological and molecular characterization of novel population of CXCR4+ SSEA-4+ Oct-4+ very small embryonic-like cells purified from human cord blood: preliminary report.

Recently, we purified from adult murine bone marrow (BM) a population of CXCR4+, Oct-4+ SSEA-1+, Sca-1+ lin− CD45− very small embryonic-like (VSEL) stem cells and hypothesized that similar cells could be also present in human cord blood (CB). Here, we report that by employing a novel two-step isolation procedure – removal of erythrocytes by hypotonic lysis combined with multiparameter sorting – we could isolate from CB a population of human cells that are similar to murine BM-derived VSELs, described previously by us. These CB-isolated VSELs (CB-VSEL) are very small (3–5 μm) and highly enriched in a population of CXCR4+AC133+CD34+lin− CD45− CB mononuclear cells, possess large nuclei containing unorganized euchromatin and express nuclear embryonic transcription factors Oct-4 and Nanog and surface embryonic antigen SSEA-4. Further studies are needed to see if human CB-isolated VSELs similar to their murine BM-derived counterparts are endowed with pluripotent stem cell properties.

A hypothesis for an embryonic origin of pluripotent Oct-4(+) stem cells in adult bone marrow and other tissues.

Accumulating evidence demonstrates that adult tissues contain a population of stem cells that express early developmental markers such as stage-specific embryonic antigen and transcription factors Oct-4 and Nanog. These are the markers characteristic for embryonic stem cells, epiblast stem cells and primordial germ cells. The presence of these stem cells in adult tissues including bone marrow, epidermis, bronchial epithelium, myocardium, pancreas and testes supports the concept that adult tissues contain some population of pluripotent stem cells that is deposited in embryogenesis during early gastrulation. In this review we will discuss these data and present a hypothesis that these cells could be direct descendants of the germ lineage. The germ lineage in order to pass genes on to the next generations creates soma and thus becomes a ‘mother lineage’ for all somatic cell lineages present in the adult body.

Cells enriched in markers of neural tissue-committed stem cells reside in the bone marrow and are mobilized into the peripheral blood following stroke.

The concept that bone marrow (BM)-derived cells participate in neural regeneration remains highly controversial and the identity of the specific cell type(s) involved remains unknown. We recently reported that the BM contains a highly mobile population of CXCR4+ cells that express mRNA for various markers of early tissue-committed stem cells (TCSCs), including neural TCSCs. Here, we report that these cells not only express neural lineage markers (β-III-tubulin, Nestin, NeuN, and GFAP), but more importantly form neurospheres in vitro. These neural TCSCs are present in significant amounts in BM harvested from young mice but their abundance and responsiveness to gradients of motomorphogens, such as SDF-1, HGF, and LIF, decreases with age. FACS analysis, combined with analysis of neural markers at the mRNA and protein levels, revealed that these cells reside in the nonhematopoietic CXCR4+/Sca-1+/lin−/CD45− BM mononuclear cell fraction. Neural TCSCs are mobilized into the peripheral blood following stroke and chemoattracted to the damaged neural tissue in an SDF-1-CXCR4−, HGF-c-Met−, and LIF-LIF-R-dependent manner. Based on these data, we hypothesize that the postnatal BM harbors a nonhematopoietic population of cells that express markers of neural TCSCs that may account for the beneficial effects of BM-derived cells in neural regeneration.

Very Small Embryonic-Like (VSEL) Stem Cells: Purification from Adult Organs, Characterization, and Biological Significance

In this review, we discuss current views of the bone marrow (BM) stem cell (SC) compartment and present data showing that BM contains heterogeneous populations of hematopoietic (H)SCs and non-HSCs. These cells are variously described in the literature as: endothelial progenitor cells (EPCs); mesenchymal (M)SCs; multipotent adult progenitor cells (MAPCs); marrow-isolated adult multilineage inducible (MIAMI) cells; and multipotent adult (MA)SCs. In some cases, it is likely that similar or overlapping populations of primitive SCs in the BM detected using various experimental strategies were assigned different names. Recently, we purified rare CXC chemokine receptor 4 expressing (CXCR4+) small SCs from the murine BM that express markers characteristic for embryonic (E)SCs, epiblast (EP)SCs, and primordial germ cells (PGCs). We named these primitive cells very small embryonic-like (VSEL) SCs. Our data indicate that VSELs may differentiate into cells from all three germ layers.

The migration of bone marrow-derived non-hematopoietic tissue-committed stem cells is regulated in an SDF-1-, HGF-, and LIF-dependent manner

Abstract.Introduction: Recently we identified in bone marrow (BM) by employing chemotactic isolation to SDF-1 gradient combined with real time RT-PCR analysis a mobile population of CXCR4+ BM mononuclear cells that express mRNA for various markers of early tissue-committed stem cells (TCSCs). In this study we evaluated whether TCSCs respond to other motomorphogens, such as hepatocyte growth factor (HGF) and leukemia inhibitory factor (LIF). Materials and Methods: We again employed chemotactic isolation combined with real-time RT-PCR analysis to assess whether murine and human BM contain TCSCs that respond to HGF and LIF gradients. We also evaluated expressions of HGF and LIF in damaged organs. Results: We noted that the number of TCSCs is highest in BM from young (1- to 2-month-old) mice and decreases in 1-year–old animals. Murine and human TCSCs 1) respond to HGF and LIF gradients in addition to an SDF-1 gradient, 2) reside in populations of BM-derived non-hematopoietic CD45−cells, and 3) are released (mobilized) from BM into the peripheral blood (PB) during tissue injury (e.g. after partial body irradiation). Conclusions: These findings further support our theory of the BM as a “hideout” for TCSCs and we suggest that their presence in BM tissue should be considered before experimental evidence is interpreted simply as transdifferentiation/plasticity of hematopoietic stem cells. Since we demonstrated that not only SDF-1, but also HGF and LIF are upregulated in damaged tissues, we postulate that CXCR4+ c-Met+ LIF-R+ TCSC could be mobilized from the BM into the PB, from which they are subsequently chemoattracted to damaged organs, where they play a role in tissue repair/regeneration.

Role of vascular endothelial growth factor (VEGF) and placenta-derived growth factor (PlGF) in regulating human haemopoietic cell growth.

Vascular endothelial growth factor (VEGF) and placental derived growth factor (PlGF) stimulate cell proliferation and differentiation by binding to their specific receptors, Flk‐1/KDR and Flt‐1 respectively. Flk‐1/KDR‐deficient murine embryos manifest failure of blood‐island formation and vasculogenesis. The aim of this study was to directly evaluate the importance of VEGF, PlGF/Flt‐1 and Flk‐1/KDR receptor ligand interactions in regulating normal and malignant human haemopoiesis. Addition of VEGF and PlGF failed to enhance survival or cloning efficiency of human haemopoietic progenitors isolated from adult bone marrows, fetal livers or cord blood samples. This finding may be explained by the apparent absence of mRNA encoding Flt‐1 and Flk‐1/KDR receptors on stem cell rich CD34+ c‐kit‐R+ Rh123low cells. Further studies revealed that Flt‐1 R mRNA, but not Flk‐1/KDR mRNA was first detectable in the more mature cells isolated from haemopoietic colonies. Accordingly, VEGF receptors are either absent, or expressed at very low level, on human haemopoietic stem/progenitor cells. Of interest, normal and malignant human haemopoietic cells appeared to secrete VEGF protein. However, in contrast to normal haemopoietic progenitors, VEGF co‐stimulated HEL cell proliferation as well as CFU‐GM colony formation from ∼15% of the chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) patients studied. Therefore, although VEGF appeared to have minimal effects on normal haemopoietic cell growth it would appear to drive malignant haemopoietic cell proliferation to some degree. Of more importance, however, we speculate that VEGF may play an very important role in leukaemogenesis by stimulating growth of vascular endothelium, thereby providing a sufficient blood supply to feed the growing haematological tumour.

Recombinant human thrombopoietin (TPO) stimulates erythropoiesis by inhibiting erythroid progenitor cell apoptosis

Thrombopoietin (TPO) has been reported to stimulate erythropoiesis, but the stimulatory mechanism has not been defined. To address this issue, we performed serum‐free cell‐culture experiments with recombinant human TPO and purified human progenitor cells. We found that TPO alone was able to stimulate the megakaryocyte colony formation in serum‐free cultures, but erythroid colonies were never observed. Only in the presence of EPO (erythropoietin) +IL‐3 was TPO able to stimulate a small increase (∼25%) in erythroid colony formation. Accordingly, we hypothesized that TPO might have an effect on erythroid progenitor cell viability, rather than a direct stimulatory effect. To test this idea, CD34+ cells were cultured for 7 d in serum‐free methylcellulose in the presence or absence of TPO, after which time KL+ EPO was added to the cultures. Cells which were pre‐cultured for 7 d in the presence of TPO gave rise to approximately 6 times as many burst forming unit‐erythroid (BFU‐E) colonies as cells which were pre‐cultured in the absence of TPO. Further, when primitive CD34+, Kit+ MNC were cultured for 3–7 d under serum‐free conditions in the presence or absence of TPO, significantly fewer cells cultured in the presence of TPO displayed apoptotic changes when compared to cells cultured in the absence of TPO. Taken together, these results suggest that TPO has little direct stimulatory effect on erythroid progenitor cells, but might indirectly enhance erythropoiesis by preventing very early erythroid progenitor cells from undergoing apoptotic cell death.

Effect of stem cell mobilization with cyclophosphamide plus granulocyte colony-stimulating factor on morphology of haematopoietic organs in mice.

Abstract.  Both granulocyte colony‐stimulating factor (G‐CSF) and cyclophosphamide (CY) are employed in the clinic as mobilizing agents to stimulate the egress of haematopoietic stem/progenitor cells (HSPC) from bone marrow (BM) into peripheral blood (PB). However, although both compounds are effective, the simultaneous administration of G‐CSF + CY allows for optimal mobilization. The aim of this study was to compare morphological changes in major haematopoietic organs in mice mobilized by G‐CSF + CY. We employed the standard G‐CSF + CY mobilization protocol, in which mice were injected at day 0 with a single dose of CY followed by daily injection of G‐CSF for 6 consecutive days. We noticed that the cytoreductive effect of CY on BM and spleen tissue was compensated at day 2 by the pro‐proliferative effect of G‐CSF. Furthermore, as evidenced by histological examination of BM sections at day 4, egress of haematopoietic cells from BM was accelerated by 2 days as compared to mobilization by G‐CSF or CY alone; also, by day 6 there was accumulation of early haematopoietic (Thy‐1low c‐kit+) cells in the spleens and livers of mobilized animals. This implies that HSPC that are mobilized from BM and circulate in PB may ‘home’ to peripheral organs. We envision that such an accumulation of these cells in the spleen (which is a major haematopoietic organ in mouse) allows them to participate in haematopoietic reconstitution. Their homing to other sites (for example the liver) is evidence that BM‐derived stem cells are playing a pivotal role in organ/tissue regeneration. The potential involvement of major chemoattractants for stem cells, like stromal‐derived factor‐1 which is induced by CY in various regenerating organs such as the liver, requires further study. We conclude that inclusion of CY into mobilization protocols on the one hand efficiently increases the egress of HSPC from the BM, but on the other hand may lead to the relocation of BM stem cell pools to peripheral tissues.

Effect of hepatocyte growth factor on early human haemopoietic cell development

Hepatocyte growth factor (HGF) stimulates cell proliferation, differentiation and migration by binding to its receptor, MET R. Whether the HGF/MET R axis plays an important regulatory role in human haemopoietic cell growth is an unresolved issue. To investigate this situation, we employed several complementary strategies including RT‐PCR, FACS analysis, and mRNA perturbation with oligodeoxynucleotides (ODN). We found that very primitive, FACS sorted, CD34+ Kit+ marrow mononuclear cells (MNC) failed to express RT‐PCR detectable MET R mRNA. In contrast, MET R expression was easily detectable by RT‐PCR in marrow stroma fibroblasts, in cells isolated from BFU‐E and CFU‐GM colonies, and in unselected normal MNC. Subsequent FACS analysis revealed that MET R protein was detectable on ∼5% of the latter cells. HGF, at concentrations of 1–50 ng/ml, had no demonstrable effect on survival or cloning efficiency of normal CD34+ MNC in serum‐free cultures. Antisense ODN mediated perturbation of MET R mRNA expression in normal CD34+ MNC, with FACS documented decline in protein expression, had no effect on the ability of these cells to give rise to haemopoietic colonies of any lineage. We also examined the biology of HGF/MET R expression in malignant haemopoietic cells. Using the strategies described above, we found that MET R mRNA was expressed in many human haemopoietic cell lines, and that the protein was expressed at high levels on HTLV transformed T lymphocytes. Wild‐type CML and AML blast cells also expressed MET mRNA, and HGF was able to co‐stimulate CFU‐GM colony formation in ∼20% of cases studied. Therefore, although the HGF/MET R axis appears to be dispensable for normal haemopoietic cell growth, it may play a role in the growth of malignant haemopoietic progenitor cells.