Pranela Rameshwar

Functional Similarities Among Genes Regulated by Oct4 in Human Mesenchymal and Embryonic Stem Cells

OCT4 is a master transcriptional regulator, which mediates pluripotency in ESCs through inhibition of tissue‐specific and promotion of stem cell‐specific genes. Suppression of OCT4, along with other regulators of pluripotency, such as SOX2 and NANOG, has been correlated with cell‐fate specification and lineage‐specific differentiation. Recent reports have shown the expression of OCT4 in adult MSCs but have not ascribed functional homology with ESCs. MSCs are mesoderm‐derived cells, primarily resident in adult bone marrow, that undergo lineage‐specific differentiation to generate specialized cells such as stroma, fat, bone, and cartilage. We have previously demonstrated the plasticity of MSCs through their ability to generate neuronal cells. Here, we show that OCT4 provides similar regulatory circuitries in human MSCs and ESCs, using chromatin immunoprecipitation‐DNA selection and ligation technology and loss‐of‐function studies. MSCs were found to express the embryonic transcription factors OCT4, NANOG, and SOX2. In addition, OCT4 was found to (a) target similar genes in MSCs and ESCs, (b) promote the expression of MSC‐specific genes, and (c) regulate MSC cell cycle progression. The results suggest similar regulatory mechanisms for OCT4 in MSCs and ESCs and have implications regarding MSC plasticity.

Specification of a Dopaminergic Phenotype from Adult Human Mesenchymal Stem Cells

Dopamine (DA) neurons derived from stem cells are a valuable source for cell replacement therapy in Parkinson disease, to study the molecular mechanisms of DA neuron development, and for screening pharmaceutical compounds that target DA disorders. Compared with other stem cells, MSCs derived from the adult human bone marrow (BM) have significant advantages and greater potential for immediate clinical application. We report the identification of in vitro conditions for inducing adult human MSCs into DA cells. Using a cocktail that includes sonic hedgehog and fibroblast growth factors, human BM‐derived MSCs were induced in vitro to become DA cells in 12 days. Based on tyrosine hydroxylase (TH) expression, the efficiency of induction was determined to be ∼67%. The cells develop a neuronal morphology expressing the neuronal markers NeuN and β III tubulin, but not glial markers, glial fibrillary acidic protein and Olig2. As the cells acquire a postmitotic neuronal fate, they downregulate cell cycle activator proteins cyclin B, cyclin‐dependent kinase 2, and proliferating cell nuclear antigen. Molecular characterization revealed the expression of DA‐specific genes such as TH, Pitx3, Nurr1, DA transporter, and vesicular monoamine transporter 2. The induced MSCs also synthesize and secrete DA in a depolarization‐independent manner. The latter observation is consistent with the low expression of voltage gated Na+ and Ca2+ channels in the induced MSCs and suggests that the cells are at an immature stage of development likely representing DA neuronal progenitors. Taken together, the results demonstrate the ability of adult human BM‐derived MSCs to form DA cells in vitro.

Brain‐derived neurotrophic factor facilitates maturation of mesenchymal stem cell‐derived dopamine progenitors to functional neurons

The generation of dopamine (DA) neurons from stem cells holds great promise in the treatment of Parkinson’s disease and other neural disease associated with dysfunction of DA neurons. Mesenchymal stem cells (MSCs) derived from the adult bone marrow show plasticity with regards to generating cells of other germ layers. In addition to reduced ethical concerns, MSCs could be transplanted across allogeneic barriers, making them desirable stem cells for clinical applications. We have reported on the generation of DA cells from human MSCs using sonic hedgehog (SHH), fibroblast growth factor 8 and basic fibroblast growth factor. Despite the secretion of DA, the cells did not show evidence of functional neurons, and were therefore designated DA progenitors. Here, we report on the role of brain‐derived neurotrophic factor (BDNF) in the maturation of the MSC‐derived DA progenitors. 9‐day induced MSCs show significant tropomyosin‐receptor‐kinase B expression, which correlate with its ligand, BDNF, being able to induce functional maturation. The latter was based on Ca2+ imaging analyses and electrophysiology. BDNF‐treated cells showed the following: increases in intracellular Ca2+ upon depolarization and after stimulation with the neurotransmitters acetylcholine and GABA and, post‐synaptic currents by electrophysiological analyses. In addition, BDNF induced increased DA release upon depolarization. Taken together, these results demonstrate the crucial role for BDNF in the functional maturation of MSC‐derived DA progenitors.

Enhancing Effect of IL-1α on Neurogenesis from Adult Human Mesenchymal Stem Cells: Implication for Inflammatory Mediators in Regenerative Medicine

Mesenchymal stem cells (MSCs) are mesoderm-derived cells, primarily resident in adult bone marrow. MSCs show lineage specificity in generating specialized cells such as stroma, fat, and cartilage. MSCs express MHC class II and function as phagocytes and APCs. Despite these immune-enhancing properties, MSCs also exert veto functions and show evidence for allogeneic transplantation. These properties, combined with ease in isolation and expansion, demonstrate MSCs as attractive candidates for tissue repair across allogeneic barriers. MSCs have also been shown to transdifferentiate in neuronal cells. We have reported expression of the neurotransmitter gene, Tac1, in MSC-derived neuronal cells, with no evidence of translation unless cells were stimulated with IL-1α. This result led us to question the potential role of immune mediators in the field of stem cell therapy. Using Tac1 as an experimental model, IL-1α was used as a prototypical inflammatory mediator to study functions on MSC-derived neuronal cells. Undifferentiated MSCs and those induced to form neurons were studied for their response to IL-1α and other proinflammatory cytokines using production of the major Tac1 peptide, substance P (SP), as readout. Although IL-1α induced high production of SP, a similar effect was not observed for all tested cytokines. The induced SP was capable of reuptake via its high-affinity NK1R and was found to stabilize IL-1R mRNA. IL-1α also enhanced the rate of neurogenesis, based on expression of neuronal markers and cRNA microarray analyses. The results provide evidence that inflammatory mediators need to be considered when deciding the course of MSC transplantation.

Dopaminergic neuronal differentiation protocol for human mesenchymal stem cells.

The generation of dopamine (DA) neurons from stem cells holds great promise for future biomedical research and in the clinical treatment of neurodegenerative diseases, such as Parkinsons disease. Mesenchymal stem cells (MSCs) derived from the adult human bone marrow (BM) can be easily isolated and expanded in culture while maintaining their immense plasticity. Here, we describe a protocol to generate DA-producing cells from adult human MSCs using a cocktail that includes sonic hedgehog (SHH), fibroblast growth factor 8 (FGF8), and basic fibroblast growth factor (bFGF). Electrophysiological functional DA neurons could be achieved by further treatment with brain-derived neurotrophic factor (BDNF). In summary, a protocol is described for the induction of primary BM-derived human MSCs to specific transdifferentiation; in this case, functional DA neurons. The MSC-derived DA cells express DA-specific markers, synthesize, and secrete dopamine. The described method could be used to generate DA cells for various model systems in which DA-producing cells are implicated in pathophysiological conditions.

Tolerance-like mediated suppression by mesenchymal stem cells in patients with dust mite allergy–induced asthma

BACKGROUND Mesenchymal stem cells (MSCs) can suppress and enhance immune functions. MSCs show promise as off-the-shelf cellular therapy for several disorders, including inflammation. OBJECTIVE We investigated the effects of MSCs on the proliferation of PBMCs to allergic subjects (dust mite [DM]), allergic asthmatic subjects, or both. METHODS Proliferation was studied by using tritiated thymidine uptake with or without MSCs. The refractoriness of PBMCs to DM was examined after preconditioning with MSCs and after repeated challenge with low-dose DM. Flow cytometry was used to study regulatory T cells and dendritic cells (DCs), and ELISA was used to study cytokine production. RESULTS Seven subjects with allergic asthma met the inclusion/exclusion criteria. MSCs significantly (P < .05) reduced the proliferation of 6 subjects with allergic asthma but not those with allergy alone. The effect was specific to the allergen because MSCs did not affect challenges to tetanus toxoid. There was no change in CD4/CD25/forkhead box protein 3-positive cells, although there were decreased IFN-γ and increased IL-10 levels. Numbers of mature DCs were increased 6-fold. Refractoriness to DM was achieved by means of repeated exposure to low-dose DM and MSCs and also MSC- preconditioned MSC. CONCLUSION MSCs suppressed the proliferation of DM-challenged PBMCs from allergic asthmatic subjects but not from allergic subjects without asthma. MSCs blunted the maturation of DCs but not regulatory T cells. Repeated exposure to low-dose DM and MSCs, as well as preconditioning of PBMCs with MSCs, caused refractoriness to DM. These findings have implications for the use of MSCs in attenuation of the inflammatory responses to allergic triggers in asthmatic patients with off-the-shelf MSCs.

Loss of RE-1 silencing factor in mesenchymal stem cell-derived dopamine progenitors induces functional maturity

Stem cell-derived dopamine (DA) neurons hold great promise for Parkinsons disease (PD). Mesenchymal stem cells (MSCs) have great potential for clinical applications. The generation of DA cells from MSCs using sonic hedgehog (SHH) and fibroblast growth factors (FGF8 and bFGF) has been reported. However, the DA cells showed weak electrical properties, representing DA neuron progenitors. Since RE-1 Silencing Factor (REST), suppresses mature neuronal genes in neuronal progenitors, we studied its role in the maturation of MSC-derived DA cells. REST expression did not change during the induction process, thus we knocked down REST and subjected MSCs to the same neural induction cocktail. We observed increases in the protein level of the Na(+) voltage-gated channel and tyrosine hydroxylase (TH). Electrophysiological analyses showed spontaneous firings and spontaneous postsynaptic currents, similar to native DA neurons. Taken together, these results show REST as the limiting gene in the generation of functional mature neurons from MSCs.

Down-Regulation of MHC II in Mesenchymal Stem Cells at High IFN-γ Can Be Partly Explained by Cytoplasmic Retention of CIITA

Mesenchymal stem cells (MSCs) are located in postnatal bone marrow, show plasticity, are linked to various bone marrow disorders, exhibit phagocytosis, exert Ag-presenting properties (APC), and are immune suppressive. Unlike professional APCs, MSCs respond bimodally to IFN-γ in MHC-II expression, with expression at 10 U/ml and baseline, and down-regulation at 100 U/ml. The effects at high IFN-γ could not be explained by down-regulation of its receptor, IFN-γRI. In this study, we report on the mechanisms by which IFN-γ regulates MHC-II expression in MSCs. Gel shift assay and Western blot analyses showed dose-dependent increases in activated STAT-1, indicating responsiveness by IFN-γRI. Western blots showed decreased intracellular MHC-II, which could not be explained by decreased transcription of the master regulator CIITA, based on RT-PCR and in situ immunofluorescence. Reporter gene assays with PIII and PIV CIITA promoters indicate constitutive expression of PIII in MSCs and a switch to PIV by IFN-γ, indicating the presence of factors for effect promoter responses. We explained decreased MHC-II at the level of transcription because CIITA protein was observed in the cytosol and not in nuclei at high IFN-γ level. The proline/serine/threonine region of CIITA showed significant decrease in phosphorylation at high IFN-γ levels. An understanding of the bimodal effects could provide insights on bone marrow homeostasis, which could be extrapolated to MSC dysfunction in hematological disorders.

Impaired erythropoiesis in the acquired immunodeficiency syndrome with disseminated Mycobacterium avium complex

PURPOSE Anemia is an important negative predictor for survival with disseminated Mycobacterium avium complex (MAC) infection in the acquired immunodeficiency syndrome (AIDS). We analyzed the differences in AIDS patients with and without MAC infection with regard to anemia, severity of human immunodeficiency virus (HIV) infection, bone marrow morphology, and bone marrow erythroid progenitor colony growth (BFU-E and CFU-E). In addition, we determined the in vitro effect of sera obtained from these patients on normal BFU-E and CFU-E. A possible role of macrophages in the suppression of erythropoiesis was examined by studying in vitro the effect of supernatants from MAC-infected macrophages on cultured BFU-E and CFU-E. PATIENTS AND METHODS Hematocrit, serum levels of p24 antigen, erythropoietin, and CD4-positive cell count were determined in 14 AIDS patients with and 24 without MAC infection. Bone marrow erythropoietic and granulocytic progenitor cells from 15 normal individuals, from 12 AIDS patients with MAC infection, and from 10 AIDS patients without MAC infection were cultured on methylcellulose. In addition, progenitor cells from normal individuals were cultured in the presence, and in the absence, of sera obtained from AIDS patients with (14), or without (24), MAC infection. Last, we studied the effect of supernatants (SNs) from MAC and Mycobacterium tuberculosis-infected macrophages on erythropoietic progenitor cell growth. RESULTS The anemia in AIDS patients with MAC infection was associated with a selective suppression of erythropoietic progenitors despite bone marrow morphology that was indistinguishable from that in patients without MAC infection. The degree of anemia could not be explained on the basis of severity of HIV infection or a deficiency of erythropoietin production. Bone marrow mononuclear cells from AIDS patients with MAC generated significantly fewer erythroid progenitor colonies (BFU-E and CFU-E) than equivalent cells from AIDS patients without MAC infection (p < 0.05). Sera from MAC-infected AIDS patients were markedly inhibitory to the erythroid progenitors as compared with sera from patients without MAC infection (p < 0.001). SNs from MAC-infected macrophages were markedly inhibitory to the erythroid progenitors (BFU-E and CFU-E) as compared with the myeloid progenitors (CFU-GM). CONCLUSION The profound anemia in MAC-infected AIDS patients is due to suppression of erythroid progenitors by a soluble factor(s) in the serum. The data suggest that the soluble factor(s) is probably elaborated by macrophages.

Moving from the Laboratory Bench to Patients’ Bedside: Considerations for Effective Therapy with Stem Cells

Although stem cell therapy is not a new field, the field was limited to transplantation of hematopoietic stem cells. Such transplantation has provided invaluable information for the emerging field with new stem cells. Mesenchymal stem cells (MSCs) are an attractive source for therapy; reduced ethical concern, ease in expansion, as off‐the‐shelf stem cells. MSCs exert immune suppressive properties, providing them with the potential for immune suppressive therapy such as autoimmunity, asthma, allergic rhinitis and graft versus host disease. In addition, MSCs, as well as other stem cells, can be applied for bone and cartilage repair, cardiovascular disease, and neural repair/protection. The data thus far with MSCs are mixed. This review discusses the immune‐enhancing properties of MSCs to explain the possible confounds of inflammatory microenvironment in the MSCs therapy. Although this review focuses on MSCs, the information can be extrapolated to other stem cells. The review summarizes the biology of MSCs, including multilineage differentiation potential, transdifferentiation capability, and immunological effects. We emphasize the key concepts that may predict the use of these cells in medicine, namely, the application of these cells from the bench to the bedside. Prospects on immunotherapy, neuroregeneration, and cardiovascular repair are used as examples of tissue repair. Clin Trans Sci 2011; Volume 4: 380–386