Cell fusion and reprogramming: resolving our transdifferences

Bone marrow-derived cells (BMDCs) can contribute to the regeneration of diverse adult tissues, including brain, liver and heart, following bone marrow transplantation. These unexpected events were initially considered a result of transdifferentiation of BMDCs, supporting the emerging idea of extended plasticity of adult stem cells. Although studies have now clearly demonstrated that spontaneous cell fusion, rather than transdifferentiation, was the primary cause for unexpected cell fate-switches of BMDCs into hepatocytes, Purkinje cells and cardiac myocytes in vivo.     

Extensive fusion of haematopoietic cells with Purkinje neurons in response to chronic inflammation

Transplanted bone marrow-derived cells (BMDCs) have been reported to fuse with cells of diverse tissues, but the extremely low frequency of fusion has led to the view that such events are biologically insignificant. Nonetheless, in mice with a lethal recessive liver disease (tyrosinaemia), transplantation of wild-type BMDCs restored liver function by cell fusion and prevented death, indicating that cell fusion can have beneficial effects. Here we report that chronic inflammation resulting from severe dermatitis or autoimmune encephalitis leads to robust fusion of BMDCs with Purkinje neurons and formation of hundreds of binucleate heterokaryons per cerebellum, a 10-100-fold higher frequency than previously reported. Single haematopoietic stem-cell transplants showed that the fusogenic cell is from the haematopoietic lineage and parabiosis experiments revealed that fusion can occur without irradiation. Transplantation of rat bone marrow into mice led to activation of dormant rat Purkinje neuron-specific genes in BMDC nuclei after fusion with mouse Purkinje neurons, consistent with nuclear reprogramming. The precise neurological role of these heterokaryons awaits elucidation, but their frequency in brain after inflammation is clearly much higher than previously appreciated.     

Role of bone marrow-derived progenitor cells in the maintenance and regeneration of dental mesenchymal tissues

While dental mesenchymal stem cells are well-studied, the origin of these cells is still unclear. Bone marrow-derived cells (BMDCs) have the potential to engraft into several tissues after injury, but whether they can become dental tissue-specific progenitor cells under normal conditions and the relationship of these cells to the tissue-resident cells are unknown. Thus, we transplanted green fluorescent protein (GFP)-labeled BMDCs into irradiated wild-type mice. We found that the engraftment of BMDCs participated in the regeneration and differentiated into periodontal specific cells after injury. Under normal conditions, there were more BMDCs engrafting into the dental mesenchymal tissue than other organs, in which the expression of stromal cell-derived factor-1 (SDF-1) was significantly higher than in other organs, and the engraftment of cells increased with time. A small fraction of GFP+ cells maintained the mesenchymal stem cell phenotype positive for CD105, CD106, and CD90, which were significantly less than the tissue-resident stem cells; meanwhile, GFP+/CD45+ cells were rare. Isolation and characterization of the dental pulp cells showed that the number of GFP+/Ki67+ cells were greater than the GFP-/Ki67+ cells. In addition, some GFP+ cells differentiated into the dental-specific cells and expressed dental-specific proteins, and can be found in the odontoblast layer after implantation of the apical bud. In conclusion, these data suggest that bone marrow progenitor cells communicate with dental tissues and become tissue-specific mesenchymal progenitor cells to maintain tissue homeostasis.     

GM-CSF induces bone marrow precursors of NOD mice to skew into tolerogenic dendritic cells that protect against diabetes

We have reported that GM-CSF treatment of NOD mice suppressed diabetes by increasing the number of tolerogenic dendritic cells (tDCs) and Tregs in the periphery. Here, we have investigated whether GM-CSF acted on NOD bone marrow DCs precursors to skew their differentiation to tDCs. DCs were generated from the bone marrow of GM-CSF-treated (GM.BMDCs) and PBS-treated (PBS.BMDCs) NOD mice and were assessed for their ability to acquire tolerogenic properties. Upon LPS stimulation, GM.BMDCs became fully mature, expressed high levels of PD-L1 and produced more IL-10 and less IL-12p70 and IFN-γ than PBS.BMDCs. In addition, LPS-stimulated GM.BMDCs possessed a reduced capacity to activate diabetogenic CD8⁺ T cells in a PD-1/PD-L1-dependent manner. A single injection of LPS-stimulated GM.BMDCs in NOD mice resulted in long-term protection from diabetes, in contrast to LPS-stimulated PBS.BMDCs. Our results showed that GM-CSF-treatment acted on bone marrow precursors to skew their differentiation into tDCs that protected NOD mice against diabetes.     

Differentiation potential and GFP labeling of sheep bone marrow‐derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are an important cell population in the bone marrow microenvironment. MSCs have the capacity to differentiate in vitro into several mesenchymal tissues including bone, cartilage, fat, tendon, muscle, and marrow stroma. This study was designed to isolate, expand, and characterize the differentiation ability of sheep bone marrow‐derived MSCs and to demonstrate the possibility to permanently express a reporter gene. Bone marrow was collected from the iliac crest and mononuclear cells were separated by density gradient centrifugation. Sheep MSCs cell lines were stable characterized as CD44+ and CD34? and then transfected with a green fluorescent protein (GFP) reporter gene. The GFP expression was maintained in about half (46.6%) of cloned blastocysts produced by nuclear transfer of GFP+ sheep MSCs, suggesting the possibility to establish multipotent embryonic cells' lines carrying the fluorescent tag for comparative studies on the differentiation capacity of adult stem cells (MSCs) versus embryonic stem cells. We found that sheep MSCs under appropriate culture conditions could be induced to differentiate into adipocytes, chondrocytes, and osteoblast lineages. Our results confirm the plasticity of sheep MSCs and establish the foundation for the development of a pre‐clinical sheep model to test the efficiency and safety of cell replacement therapy. J. Cell. Biochem. 114: 134–143, 2012. © 2012 Wiley Periodicals, Inc.     

Selective inactivation or reconstitution of adenosine A2A receptors in bone marrow cells reveals their significant contribution to the development of ischemic brain injury

Inactivation of the adenosine A(2A) receptor (A(2A)R) consistently protects against ischemic brain injury and other neural insults, but the relative contribution of A(2A)Rs on peripheral inflammatory cells versus A(2A)Rs expressed on neurons and glia is unknown. We created a chimeric mouse model in which A(2A)Rs on bone marrow-derived cells (BMDCs) were selectively inactivated or reconstituted by bone marrow transplantation. Selective reconstitution of A(2A)Rs on BMDCs (A(2A)R knockout mice transplanted with wild-type bone marrow cells) largely reinstates ischemic brain injury in global A(2A)R knockout mice. Conversely, selective inactivation of A(2A)Rs on BMDCs (wild-type mice transplanted with A(2A)R knockout bone marrow cells) attenuates infarct volumes and ischemia-induced expression of several proinflammatory cytokines in the brain, but exacerbates ischemic liver injury. These results indicate that the A(2A)R-stimulated cascade in BMDCs is an important modulator of ischemic brain injury and that ischemic brain and liver injuries are regulated distinctly by A(2A)Rs on BMDCs.     

Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue.

Human mesenchymal stem cells (MSC), that have been reported to be present in bone marrow, adipose tissues, dermis, muscles and peripheral blood, have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle and neuron. This differentiation potential makes MSC excellent candidates for cell-based tissue engineering. In this study, we have examined phenotypes and gene expression profile of the human adipose tissue-derived stromal cells (ATSC) in the undifferentiated states, and compared with that of bone marrow stromal cells (BMSC). ATSC were enzymatically released from adipose tissues from adult human donors and were expanded in monolayer with serial passages at confluence. BMSC were harvested from the metaphysis of adult human femur. Flowcytometric analysis showed that ATSC have a marker expression that is similar to that of BMSC. ATSC expressed CD29, CD44, CD90, CD105 and were absent for HLA-DR and c-kit expression. Under appropriate culture conditions, MSC were induced to differentiate to the osteoblast, adipocyte, and chondrogenic lineages. ATSC were superior to BMSC in respect to maintenance of proliferating ability, and microarray analysis of gene expression revealed differentially expressed genes between ATSC and BMSC. The proliferating ability and differentiation potential of ATSC were variable according to the culture condition. The similarities of the phenotypes and the gene expression profiles between ATSC and BMSC could have broad implications for human tissue engineering.     

High-resolution in-vivo analysis of normal brain response to cranial irradiation

Radiation therapy (RT) is a widely accepted treatment strategy for many central nervous system (CNS) pathologies. However, despite recognized therapeutic success, significant negative consequences are associated with cranial irradiation (CR), which manifests months to years post-RT. The pathophysiology and molecular alterations that culminate in the long-term detrimental effects of CR are poorly understood, though it is thought that endothelial injury plays a pivotal role in triggering cranial injury. We therefore explored the contribution of bone marrow derived cells (BMDCs) in their capacity to repair and contribute to neo-vascularization following CR. Using high-resolution in vivo optical imaging we have studied, at single-cell resolution, the spatio-temporal response of BMDCs in normal brain following CR. We demonstrate that BMDCs are recruited specifically to the site of CR, in a radiation dose and temporal-spatial manner. We establish that BMDCs do not form endothelial cells but rather they differentiate predominantly into inflammatory cells and microglia. Most notably we provide evidence that more than 50% of the microglia in the irradiated region of the brain are not resident microglia but recruited from the bone marrow following CR. These results have invaluable therapeutic implications as BMDCs may be a primary therapeutic target to block acute and long-term inflammatory response following CR. Identifying the critical steps involved in the sustained recruitment and differentiation of BMDCs into microglia at the site of CR can provide new insights into the mechanisms of injury following CR offering potential therapeutic strategies to counteract the long-term adverse effects of CR.     

Rosmarinic acid down-regulates the LPS-induced production of monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) via the MAPK pathway in bone-marrow derived dendritic cells

In the present study, we investigated whether rosmarinic acid, which has been suggested to exhibit anti-inflammatory properties, can suppress the expressions of monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) via the MAPK pathway in LPS-stimulated bone marrow-derived dendritic cells (BMDCs) in the presence of GM-CSF and IL-4 in media. The effects of rosmarinic acid were investigated in BMDCs with respect to the following; cytotoxicity, surface molecule expression, dextran-FITC uptake, cell migration, chemokine gene expression, and the MAPK signaling pathway. Rosmarinic acid was found to significantly inhibit the expressions of CD80, CD86, MHC class I, and MHC class II in LPS-stimulated mature BMDCs, and rosmarinic acid-treated BMDCs were found to be highly efficient with regards to antigen capture via mannose receptor-mediated endocytosis. In addition, rosmarinic acid reduced cell migration by inducing the expression of a specific chemokine receptor on LPS-induced mature BMDCs. Rosmarinic acid also significantly reduced the expressions of MCP-1 and MIP-lalpha induced by LPS in BMDCs and inhibited LPS-induced activation of MAPK and the nuclear translocation of NF-kappaB. These findings broaden current perspectives concerning our understanding of the immunopharmacological functions of rosmarinic acid, and have ramifications that concern the development of therapeutic drugs for the treatment of DC-related acute and chronic diseases.     

Adeno-associated virus type 2-mediated transduction of murine hematopoietic cells with long-term repopulating ability and sustained expression of a human globin gene in vivo.

Adeno-associated virus type 2 (AAV), a nonpathogenic human parvovirus, is gaining attention as a vector for potential use in human gene therapy. We and others have described AAV-mediated beta-globin gene transfer and expression in established human and murine erythroleukemia cell lines in vitro. However, successful AAV-mediated globin gene transduction of hematopoietic stem cells and long-term expression in vivo in progeny cells have not been documented. We report here that infection of murine hematopoietic bone marrow cells ex vivo with a recombinant AAV vector containing the genomic copy of a normal human globin gene followed by transplantation of these cells into lethally irradiated congenic mice resulted in efficient gene transfer into hematopoietic cells with long-term repopulating ability as detected by the presence of the human globin gene sequences in bone marrow and spleen in primary recipient mice for at least 6 months. Long-term expression of the human globin gene was also detected in bone marrow, but not in spleen, in primary recipient mice. Furthermore, in secondary-transplant experiments, we were also able to document the presence as well as expression of the transduced human globin gene in mouse bone marrow for up to 3 months. These results provide further support for potential use of the AAV-based vector system in gene therapy of human hemoglobinopathies in general and sickle-cell anemia and beta-thalassemia in particular.     

Comparative osteogenic transcription profiling of various fetal and adult mesenchymal stem cell sources

Human mesenchymal stem cells (MSC) from adult and fetal tissues are promising candidates for cell therapy but there is a need to identify the optimal source for bone regeneration. We have previously characterized MSC populations in first trimester fetal blood, liver, and bone marrow and we now evaluate their osteogenic differentiation potential in comparison to adult bone marrow MSC. Using quantitative real-time RT-PCR, we demonstrated that 16 osteogenic-specific genes (OC, ON, BSP, OP, Col1, PCE, Met2A, OPG, PHOS1, SORT, ALP, BMP2, CBFA1, OSX, NOG, IGFII) were expressed in both fetal and adult MSC under basal conditions and were up-regulated under osteogenic conditions both in vivo and during an in vitro 21-day time-course. However, under basal conditions, fetal MSC had higher levels of osteogenic gene expression than adult MSC. Upon osteogenic differentiation, fetal MSC produced more calcium in vitro and reached higher levels of osteogenic gene up-regulation in vivo and in vitro. Second, we observed a hierarchy within fetal samples, with fetal bone marrow MSC having greater osteogenic potential than fetal blood MSC, which in turn had greater osteogenic potential than fetal liver MSC. Finally, we found that the level of gene expression under basal conditions was positively correlated with both calcium secretion and gene expression after 21 days in osteogenic conditions. Our findings suggest that stem cell therapy for bone dysplasias such as osteogenesis imperfecta may benefit from preferentially using first trimester fetal blood or bone marrow MSC over fetal liver or adult bone marrow MSC.     

Nestin Positive Bone Marrow Derived Cells Responded to Injury Mobilize into Peripheral Circulation and Participate in Skin Defect Healing

Exogenously infused mesenchymal stem cells (MSCs) are thought to migrate to injury site through peripheral blood stream and participate in tissue repair. However, whether and how endogenous bone marrow MSCs mobilized to circulating and targeted to tissue injury has raised some controversy, and related studies were restricted by the difficulty of MSCs identifying in vivo. Nestin, a kind of intermediate filament protein initially identified in neuroepithelial stem cells, was recently reported as a credible criteria for MSCs in bone marrow. In this study, we used a green fluorescent protein (GFP) labeled bone marrow replacement model to trace the nestin positive bone marrow derived cells (BMDCs) of skin defected-mice. We found that after skin injured, numbers of nestin⁺ cells in peripheral blood and bone marrow both increased. A remarkable concentration of nestin⁺ BMDCs around skin wound was detected, while few of these cells could be observed in uninjured skin or other organs. This recruitment effect could not be promoted by granulocyte colony-stimulating factor (G-CSF), suggests a different mobilization mechanism from ones G-CSF takes effect on hematopoietic cells. Our results proposed nestin⁺ BMDCs as mobilized candidates in skin injury repair, which provide a new insight of endogenous MSCs therapy.     

Mesenchymal stem cells: characteristics and clinical applications

Mesenchymal stem cells (MSCs) are bone marrow populating cells, different from hematopoietic stem cells, which possess an extensive proliferative potential and ability to differentiate into various cell types, including: osteocytes, adipocytes, chondrocytes, myocytes, cardiomyocytes and neurons. MSCs play a key role in the maintenance of bone marrow homeostasis and regulate the maturation of both hematopoietic and non-hematopoietic cells. The cells are characterized by the expression of numerous surface antigens, but none of them appears to be exclusively expressed on MSCs. Apart from bone marrow, MSCs are located in other tissues, like: adipose tissue, peripheral blood, cord blood, liver and fetal tissues. MSCs have been shown to be powerful tools in gene therapies, and can be effectively transduced with viral vectors containing a therapeutic gene, as well as with cDNA for specific proteins, expression of which is desired in a patient. Due to such characteristics, the number of clinical trials based on the use of MSCs increase. These cells have been successfully employed in graft versus host disease (GvHD) treatment, heart regeneration after infarct, cartilage and bone repair, skin wounds healing, neuronal regeneration and many others. Of special importance is their use in the treatment of osteogenesis imperfecta (OI), which appeared to be the only reasonable therapeutic strategy. MSCs seem to represent a future powerful tool in regenerative medicine, therefore they are particularly important in medical research.     

Evaluation of Tat-encoding bicistronic human immunodeficiency virus type 1 gene transfer vectors in primary canine bone marrow mononuclear cells

Tat-encoding human immunodeficiency virus type 1 (HIV-1) gene transfer vectors were evaluated in primary canine bone marrow mononuclear cells. Tat vectors provided higher levels of gene expression than vectors with internal promoters. The HIV-1 vector was also more efficient than Moloney murine leukemia virus (MoMLV) vectors for transduction of canine bone marrow mononuclear cells in vitro. Transplantation experiments in dogs with transduced autologous marrow cells confirmed the superiority of HIV-1 vectors over MoMLV vectors for gene transfer into canine bone marrow cells. Tat vectors may be useful not only for providing high levels of therapeutic gene expression in hematopoietic cells but also for study of the biological effects of Tat in those tissues in the canine model.     

Antitumor effects of a xenogeneic survivin bone marrow derived dendritic cell vaccine against murine GL261 gliomas

Survivin is a member of the inhibitor of apoptosis protein family. Gliomas and many other tumors express survivin at high levels; whereas, normal fully differentiated cells generally do not. Therefore, survivin represents a tumor-specific target for cancer vaccine therapy. It has been shown that it is possible to produce a MHC-I-restricted cellular immunologic response to survivin vaccines. To study differences in immunogenicity between murine and human survivin proteins, we vaccinated C57BL/6 mice with bone marrow dendritic cells (BMDC) transfected with expression vectors containing the murine and human survivin genes. Mice vaccinated with BMDCs expressing a truncated human survivin protein developed cytotoxic T lymphocyte to subcutaneous GL261 glioma cells and exhibited prolonged tumor-free survival compared to mice vaccinated with BMDCs transfected with vector alone (P<0.01). While mice challenged with intracerebral GL261 cells had increased survival, no cures were observed. In contrast, vaccinated mice that fully resisted subcutaneous tumor challenge were rendered resistant to intracerebral GL261 re-challenge. BMDCs transfected with the full-length human survivin molecule were significantly more effective at prolonging survival than BMDCs expressing the full-length murine survivin gene (P=0.0175). Therefore, xenogeneic differences between human and murine sequences might be exploited to develop more immunogenic tumor vaccines.     

Reduced lymphocyte longevity and homeostatic proliferation in lamin B receptor-deficient mice results in profound and progressive lymphopenia

The lamin B receptor (LBR) is a highly unusual inner nuclear membrane protein with multiple functions. Reduced levels are associated with decreased neutrophil lobularity, whereas complete absence of LBR results in severe skeletal dysplasia and in utero/perinatal lethality. We describe a mouse pedigree, Lym3, with normal bone marrow and thymic development but profound and progressive lymphopenia particularly within the T cell compartment. This defect arises from a point mutation within the Lbr gene with only trace mutant protein detectable in homozygotes, albeit sufficient for normal development. Reduced T cell homeostatic proliferative potential and life span in vivo were found to contribute to lymphopenia. To investigate the role of LBR in gene silencing in hematopoietic cells, we examined gene expression in wild-type and mutant lymph node CD8 T cells and bone marrow neutrophils. Although LBR deficiency had a very mild impact on gene expression overall, for common genes differentially expressed in both LBR-deficient CD8 T cells and neutrophils, gene upregulation prevailed, supporting a role for LBR in their suppression. In summary, this study demonstrates that LBR deficiency affects not only nuclear architecture but also proliferation, cell viability, and gene expression of hematopoietic cells.     

Chemically-induced cancers do not originate from bone marrow-derived cells

Background

The identification and characterization of cancer stem cells (CSCs) is imperative to understanding the mechanism of cancer pathogenesis. Growing evidence suggests that CSCs play critical roles in the development and progression of cancer. However, controversy exists as to whether CSCs arise from bone marrow-derived cells (BMDCs).

Methodology and Principal Findings

In the present study, n-nitrosodiethylamine (DEN) was used to induce tumor formation in female mice that received bone marrow from male mice. Tumor formation was induced in 20/26 mice, including 12 liver tumors, 6 lung tumors, 1 bladder tumor and 1 nasopharyngeal tumor. Through comparison of fluorescence in situ hybridization (FISH) results in corresponding areas from serial tumor sections stained with H&E, we determined that BMDCs were recruited to both tumor tissue and normal surrounding tissue at a very low frequency (0.2–1% in tumors and 0–0.3% in normal tissues). However, approximately 3–70% of cells in the tissues surrounding the tumor were BMDCs, and the percentage of BMDCs was highly associated with the inflammatory status of the tissue. In the present study, no evidence was found to support the existence of fusion cells formed form BMDCs and tissue-specific stem cells.

Conclusions

In summary, our data suggest that although BMDCs may contribute to tumor progression, they are unlike to contribute to tumor initiation.     

Preferential expression of the c-fps protein in chicken macrophages and granulocytic cells.

We have studied the expression of the protein kinase activity of NCP98, the c-fps gene product, in several hemopoietic tissues of chickens as a function of the developmental stage of these organs. We found that in bone marrow, spleen, and bursa, maximum NCP98 kinase activity on a per-cell basis correlates with the peak of granulopoiesis in these organs. Furthermore, in a bovine serum albumin density gradient fractionation of bone marrow cells, granulocytic cells appeared to account for most of the NCP98 kinase activity. No correlation was found between the distribution of erythrocytic, lymphocytic, or thrombocytic cells and the distribution of the expression of NCP98 kinase activity. However, NCP98 protein and kinase activity were 10-fold higher in macrophages than in bone marrow. In addition, depletion by complement-mediated lysis of erythrocytic cells in bone marrow did not significantly reduce the total recovery of NCP98 kinase activity. These results argue for the specific expression of the c-fps gene product in granulocytic cells and macrophages.