Ferenc Uher

Mesenchymal Stem Cells Cooperate with Bone Marrow Cells in Therapy of Diabetes

Several recent studies have suggested that the adult bone marrow harbors cells that can influence β‐cell regeneration in diabetic animals. Other reports, however, have contradicted these findings. To address this issue, we used an animal model of type 1 diabetes in which the disease was induced with streptozotocin in mice. Freshly prepared sex‐mismatched bone marrow cells (BMCs) and syngeneic or allogeneic mesenchymal stem cells (MSCs) were concomitantly administrated into sublethally irradiated diabetic mice. Blood glucose and serum insulin concentrations rapidly returned to normal levels, accompanied by efficient tissue regeneration after a single injection of a mixture of 106 BMCs per 105 MSCs. Neither BMC nor MSC transplantation was effective alone. Successful treatment of diabetic animals was not due to the reconstitution of the damaged islet cells from the transplant, since no donor‐derived β‐cells were found in the recovered animals, indicating a graft‐initiated endogenous repair process. Moreover, MSC injection caused the disappearance of β‐cell‐specific T lymphocytes from diabetic pancreas. Therefore, we suggest that two aspects of this successful treatment regimen operate in parallel and synergistically in our model. First, BMCs and MSCs induce the regeneration of recipient‐derived pancreatic insulin‐secreting cells. Second, MSCs inhibit T‐cell‐mediated immune responses against newly formed β‐cells, which, in turn, are able to survive in this altered immunological milieu. Thus, the application of this therapy in human patients suffering from diabetes and/or other tissue destructive autoimmune diseases may be feasible.

Biphasic Effect of Recombinant Galectin‐1 on the Growth and Death of Early Hematopoietic Cells

Galectin‐1 is a member of the family of β‐galactoside binding animal lectins, galectins. Its presence in the bone marrow has been detected; however, its role in the regulation of hematopoiesis is unknown. In the present study, we have evaluated the effect of recombinant human galectin‐1 on the proliferation and survival of murine and human hematopoietic stem and progenitor cells. We show that low amount of galectin‐1 (10 ng/ml) increases the formation of granulocyte‐macrophage and erythroid colonies and the frequencies of day‐7 cobblestone area–forming cells on a lactose‐inhibitable fashion. In contrast, high amount of galectin‐1 (10 μg/ml) dramatically reduces the growth of the committed blood‐forming progenitor cells as well as the much younger, lineage‐negative hematopoietic cells (day‐28 to −35 cobblestone area–forming cells). This inhibition is not blocked by lactose and, therefore, is largely independent of the β‐galactoside–binding site of the lectin. Furthermore, assays to detect apoptosis render it likely that the high amount of galectin‐1 acts as a classical proapoptotic factor for the premature hematopoietic cells.

Mechanism of tumor cell-induced T-cell apoptosis mediated by galectin-1.

Galectin-1 (Gal-1) has been implicated in tumor progression partly via the induction of T-cell apoptosis. However the mechanism of Gal-1 induced T-cell death was mostly studied using recombinant, soluble Gal-1 producing controversial results. To explore the true mechanism of Gal-1 and hence tumor cell-induced T-cell death, we applied co-cultures of tumor cells and T-cells thus avoiding artificial circumstances generated using recombinant protein. T-cells died when co-cultured with Gal-1-expressing but survived with Gal-1 non-expressing tumor cells. Removing tumor cell surface Gal-1 or knocking down Gal-1 expression resulted in diminution of T-cell apoptosis. Gal-1 transgenic or soluble Gal-1 treated HeLa cells became cytotoxic. Stimulation of apoptosis required interaction between the tumor and T-cells, presence of p56lck and ZAP70, decrease of mitochondrial membrane potential and caspase activation. Hence tumor cell-derived Gal-1 might efficiently contribute to tumor self-defense. Moreover this system resolves the discrepancies obtained using recombinant Gal-1 in T-cell apoptosis studies.

Soluble Jagged-1 is able to inhibit the function of its multivalent form to induce hematopoietic stem cell self-renewal in a surrogate in vitro assay

Stem cells reside in customized microenvironments (niches) that contribute to their unique ability to divide asymmetrically to give rise to self and to a daughter cell with distinct properties. Notch receptors and their ligands are highly conserved and have been shown to regulate cell‐fate decisions in multiple developmental systems through local cell interactions. To assess whether Notch signaling may regulate hematopoiesis to maintain cells in an immature state, we examined the functional role of the recombinant, secreted form of the Notch ligand Jagged‐1 during mouse hematopoietic stem cell (HSC) and progenitor cell proliferation and maturation. We found that ligand immobilization on stromal layer or on Sepharose‐4B beads is required for the induction of self‐renewing divisions of days 28–35 cobblestone area‐forming cell. The free, soluble Jagged‐1, however, has a dominant‐negative effect on self‐renewal in the stem‐cell compartment. In contrast, free as well as immobilized Jagged‐1 promotes growth factor‐induced colony formation of committed hematopoietic progenitor cells. Therefore, we propose that differences in Jagged‐1 presentation and developmental stage of the Notch receptor‐bearing cells influence Notch ligand‐binding results toward activation or inhibition of downstream signaling. Moreover, these results suggest potential clinical use of recombinant Notch ligands for expanding human HSC populations in vitro.

Activated T-cells and pro-inflammatory cytokines differentially regulate prostaglandin E2 secretion by mesenchymal stem cells

In recent years it has become clear that mesenchymal stem or stromal cells (MSCs) are capable of modulating inflammatory and immune responses through interaction with a wide variety of cells. Whereas several studies indicated that PGE2 is one of the chief soluble mediators involved in these processes, here we investigated prostaglandin E2 (PGE2) production of murine bone marrow- (BM-) and adipose tissue- (Ad-) derived MSCs stimulated with pro-inflammatory cytokines TNF-α and IFN-γ, or co-cultured with ConA-induced T-cell blasts. We found that both MSC populations are able to produce high amounts of PGE2 in MSC/activated T-cell co-cultures. This effect was markedly attenuated when direct cell-cell contact was prevented in transwell system, indicating that the elicitation of the PGE2 secretion of MSCs is contact-dependent in this experimental setting. In contrast, when soluble recombinant pro-inflammatory cytokines were added to the MSC cultures, TNF-α and IFN-γ act synergistically to induce PGE2 production, whereas only high amount of TNF-α but not IFN-γ was able to do so alone. Although the PGE2 secretion by MSCs was completely abrogated by addition of indomethacin under all culture conditions tested, L-NMA, a NOS inhibitor could only partially inhibit it when the cells were elicited in the concomitant presence of TNF-α and IFN-γ. These results, combined with others, suggest that NO acts downstream of IFN-γ but upstream of COX2. Taken together, our findings demonstrate that the induction of PGE2 secretion by BM- and Ad-MSCs is not mediated by a single or unique, nonredundant molecular mechanism under different experimental conditions.

Chondrogenic Potential of Mesenchymal Stem Cells from Patients with Rheumatoid Arthritis and Osteoarthritis: Measurements in a Microculture System

Background: Mesenchymal stem cells (MSCs) have the potential to differentiate into distinct mesenchymal tissues; including cartilage and bone, they can be an attractive cell source for cartilage tissue engineering approaches. Our objective here was to compare the in vitro chondrogenic potential of MSCs isolated from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) with cells from normal donors. Methods: Marrow samples were removed during bone surgery and adherent cell cultures were established. The cells were then passed into a newly developed microaggregate culture system in a medium containing transforming growth factor β3, insulin, dexamethasone and/or demineralized bone matrix. In vitro chondrogenic activity was measured as metabolic sulfate incorporation and type II collagen expression in pellet cultures. Results: Culture-expanded MSCs from RA and OA patients did not differ significantly from the normal population with respect to their chondrogenic potential in vitro. Capability of total protein and proteoglycan synthesis as well as collagen II mRNA expression by cell aggregates was similar for all cell preparations in the presence of the appropriate growth and differentiation factors. Chondroprotective drugs such as chondroitin sulfate and glucosamine enhanced, whereas chloroquine inhibited chondrogenesis in normal donor-derived or patient-derived MSC cultures. Galectin-1, a β-galactoside-binding protein with marked anti-inflammatory activity, stimulated the chondrogenic differentiation of mesenchymal cells in low (<2 μg/ml) concentration. Discussion: These findings show that MSCs from RA and OA patients possess similar chondrogenic potential as MSCs isolated from healthy donors, therefore these cells may serve as a potential new prospect in cartilage replacement therapy.

Inappropriate notch activity and limited mesenchymal stem cell plasticity in the bone marrow of patients with myelodysplastic syndromes

Myelodysplastic syndromes (MDSs) are a heterogeneous group of hematological disorders characterized by ineffective hematopoiesis, enhanced bone marrow apoptosis and frequent progression to acute myeloid leukemia. Several recent studies suggested that, besides the abnormal development of stem cells, microenvironmental alterations are also present in the MDS bone marrow. In this study, we have examined the relative frequencies of stem and progenitor cell subsets of MDS and normal hematopoietic cells growing on stromal cell layers established from MDS patients and from normal donors. When hematopoietic cells from MDS patients were co-cultured with normal stromal cells, the frequency of either early or late cobblestone area-forming cells (CAFC) was significantly lower compared to the corresponding normal control values in 4 out of 8 patients. In the opposite situation, when normal hematopoietic cells were incubated on MDS stromal cells, the CAFC frequencies were decreased in 5 out of 6 patients, compared to normal stromal layer-containing control cultures. Moreover, a soluble Notch ligand (Jagged-1 protein) was an inhibitor of day-35-42 CAFC when normal hematopoietic cells were cultured with normal or MDS stromal cells, but was unable to inhibit MDS stem and early progenitor cell growth (day-35-42 CAFC) on pre-established stromal layers. These findings suggest that in early hematopoietic cells isolated from MDS patients the Notch signal transduction pathway is disrupted. Furthermore, there was a marked reduction in the plasticity of mesenchymal stem cells of MDS patients compared with those of normal marrow donors, in neurogenic and adipogenic differentiation ability and hematopoiesis supporting capacityin vitro. These results are consistent with the hypothesis that when alterations are present in the myelodysplastic stroma environment along with intrinsic changes in a hematopoietic stem/progenitor cell clone, both factors might equally contribute to the abnormal hematopoiesis in MDS.

Identical, similar or different? Learning about immunomodulatory function of mesenchymal stem cells isolated from various mouse tissues: bone marrow, spleen, thymus and aorta wall

Mesenchymal stem or multipotent stromal cells (MSCs) have been implicated in tissue maintenance and repair and regulating immune effector cells through different mechanisms. These functions in mouse were primarily described for bone marrow (BM)-derived MSCs. To learn more about MSCs of different tissue origin, we compared the immunophenotype, differentiation ability to adipocyte and bone and immunomodulatory activity of MSCs isolated from BM, spleen, thymus and aorta wall of 14-day-old C57Bl/6 mice. The established cell lines fulfilled the requirements described for MSCs in terms of morphology, surface marker expression and differentiation potential although they were distinguishable regarding the expression pattern of the MSC markers and ability generating other cell types. Most importantly, a remarkable diversity was shown in the capacity of inhibition of mitogen- and alloantigen-induced T-cell proliferation, since BM- and spleen-derived MSCs were the most powerful aorta-derived MSCs were less effective, whereas thymus-derived mesenchymal cells were unable to block T-cell growth in vitro. Accordingly, BM, spleen and aorta, but not thymus-derived MSCs, in combination with BM hematopoietic cells were equally efficient to prevent streptozotocin-induced diabetes in vivo. These findings suggested that MSCs residing in different organs might stem from common ancestor; however, once populating into a given tissue microenvironment, they acquire specific properties mainly in the term of the immunoregulatory function.

In vitro effect of carboplatin, cytarabine, paclitaxel, vincristine, and low-power laser irradiation on murine mesenchymal stem cells

Mesenchymal stem cells (MSCs) are promising for use in regenerative medicine. Cytostatics can decrease, but low‐power laser irradiation (LPLI) can increase the growth of MSCs. The interaction of LPLI, MSCs and cytostatics is not known. This study investigated the effect of four cytostatics (carboplatin, cytarabine, paclitaxel, vincristine), LPLI, and combination of a cytostatic drug and LPLI on murine MSCs (mMSCs).

Detection of drug-induced apoptosis by flow cytometry after alkaline extraction of ethanol fixed cells

A new flow cytometric method was developed to detect apoptotic cells with fragmented DNA and to determine cell cycle distribution of viable cells, in the same sample, by propidium iodide staining. Apoptosis, in HT58 human B lymphoma cells, was induced by etoposide and/or by staurosporine. Using appropriate alkaline solutions (between 1-10 mN NaOH in 150 mM saline) followed by neutralization with buffer solution, the fragmented DNA can be extracted quantitatively from ethanol fixed cells. Further, good resolution of the cell cycle distribution can be obtained in unimpaired cells without RNase treatment. Furthermore, unlike the widely used hypotonic-detergent extraction of unfixed cells, the suggested extraction method can prevent drug-induced disintegration of dead cells when karyorrhexis occurs.