Mouse Bone Marrow-derived Cells Research Articles

Osteoclastic cell behaviors affected by the α-tricalcium phosphate based bone cements

Calcium phosphate cements (CPCs) have recently gained great interest as injectable bone substitutes for use in dentistry and orthopedics. α-tricalcium phosphate (α-TCP) is a popularly used precursor powder for CPCs. When mixed with appropriate content of liquid and kept under aqueous conditions, α-TCP dissolves to form a calcium-deficient hydroxyapatite and then hardens to cement. In this study, α-TCP based cement (CP) and its composite cement with chitosan (Ch-CP) were prepared and the osteoclastic responses to the cements and their elution products were evaluated. Preliminary evaluation of the cements revealed that the CP and Ch-CP hardened within ~10 min at an appropriate powder-to-liquid ratio (PL) of 3.0. In addition, CP and Ch-CP were transformed into an apatite phase following immersion in a saline solution. Moreover, the osteoblastic cells were viable on the cements for up to 10 days. Mouse-derived bone marrow cells were isolated and activated with osteoclastic differentiation medium, and the effects of the CP and Ch-CP substrates and their ionic eluants on the osteoclastic activity were investigated. Osteoclastic cells were viable for up to 14 days on both types of cements, maintaining a higher cell growth level than the control culture dish. Multi-nucleated osteoclastic cells that were tartrate-resistant acid phosphatase (TRAP)-positive were clearly observed when cultured on the cement substrates as well as treated with the cement eluants. The TRAP activity was found to be significantly higher in cells influenced by the cement substrates and their eluants with respect to the control culture dish (Ch-CP > CP ≫ control). Overall, the osteoclastic differentiation was highly stimulated by the α-TCP based experimental cements in terms of both the substrate interaction and their elution products.

The Peptide Sequence of Diacyl Lipopeptides Determines Dendritic Cell TLR2-Mediated NK Activation

Natural killer (NK) cells are lymphocyte effectors that are activated to control certain microbial infections and tumors. Many NK-activating and regulating receptors are involved in regulating NK cell function. In addition, activation of naïve NK cells is fundamentally triggered by cytokines or myeloid dendritic cells (mDC) in various modes. In this study, we synthesized 16 S-[2,3-bis(palmitoyl)propyl]cysteine (Pam2Cys) lipopeptides with sequences designed from lipoproteins of Staphylococcus aureus, and assessed their functional properties using mouse (C57BL/6) bone marrow-derived DC (BMDC) and NK cells. NK cell activation was evaluated by three criteria: IFN-γ production, up-regulation of NK activation markers and cytokines, and NK target (B16D8 cell) cytotoxicity. The diacylated lipopeptides acted as TLR2 ligands, inducing up-regulation of CD25/CD69/CD86, IL-6, and IL-12p40, which represent maturation of BMDC. Strikingly, the Pam2Cys lipopeptides induced mouse NK cell activation based on these criteria. Cell-cell contact by Pam2Cys peptide-stimulated BMDC and NK cells rather than soluble mediators released by stimulated BMDC induced activation of NK cells. For most lipopeptides, the BMDC TLR2/MyD88 pathway was responsible for driving NK activation, while some slightly induced direct activation of NK cells via the TLR2/MyD88 pathway in NK cells. The potential for NK activation was critically regulated by the peptide primary sequence. Hydrophobic or proline-containing sequences proximal to the N-terminal lipid moiety interfered with the ability of lipopeptides to induce BMDC-mediated NK activation. This mode of NK activation is distinctly different from that induced by polyI:C, which is closely associated with type I IFN-inducing pathways of BMDC. These results imply that the MyD88 pathway of BMDC governs an alternative NK-activating pathway in which the peptide sequence of TLR2-agonistic lipopeptides critically affects the potential for NK activation.

PPARα Is Essential for Microparticle-Induced Differentiation of Mouse Bone Marrow-Derived Endothelial Progenitor Cells and Angiogenesis

BackgroundBone marrow-derived endothelial progenitor cells (EPCs) are critical for neovascularization. We hypothesized that microparticles (MPs), small fragments generated from the plasma membrane, can activate angiogenic programming of EPCs.Methodology/Principal FindingsWe studied the effects of MPs obtained from wild type (MPsPPARα+/+) and knock-out (MPsPPARα−/−) mice on EPC differentiation and angiogenesis. Bone marrow-derived cells were isolated from WT or KO mice and were cultured in the presence of MPsPPARα+/+ or MPsPPARα−/− obtained from blood of mice. Only MPsPPARα+/+ harboring PPARα significantly increased EPC, but not monocytic, differentiation. Bone marrow-derived cells treated with MPsPPARα+/+ displayed increased expression of pro-angiogenic genes and increased in vivo angiogenesis. MPsPPARα+/+ increased capillary-like tube formation of endothelial cells that was associated with enhanced expressions of endothelial cell-specific markers. Finally, the effects of MPsPPARα+/+ were mediated by NF-κB-dependent mechanisms.Conclusions/SignificanceOur results underscore the obligatory role of PPARα carried by MPs for EPC differentiation and angiogenesis. PPARα-NF-κB-Akt pathways may play a pivotal stimulatory role for neovascularization, which may, at least in part, be mediated by bone marrow-derived EPCs. Improvement of EPC differentiation may represent a useful strategy during reparative neovascularization.

Cilostazol enhances neovascularization in the mouse hippocampus after transient forebrain ischemia

Cilostazol is known to be a specific type III phosphodiesterase inhibitor, which promotes increased intracellular cAMP levels. We assessed the effect of cilostazol on production of angioneurins and chemokines and recruitment of new endothelial cells for vasculogenesis in a mouse model of transient forebrain ischemia. Pyramidal cell loss was prominently evident 3-28 days postischemia, which was markedly ameliorated by cilostazol treatment. Expression of angioneurins, including endothelial nitric oxide synthase, vascular endothelial growth factor, and brain-derived neurotrophic factor, was up-regulated by cilostazol treatment in the postischemic hippocampus. Cilostazol also increased Sca-1/vascular endothelial growth factor receptor-2 positive cells in the bone marrow and circulating peripheral blood and the number of stromal cell-derived factor-1alpha-positive cells in the molecular layer of the hippocampus, which colocalized with CD31. CXCR4 chemokine receptors were up-regulated by cilostazol in mouse bone marrow-derived endothelial progenitor cells, suggesting that cilostazol may be important in targeting or homing in of bone marrow-derived stem cells to areas of injured tissues. CD31-positive cells were colocalized with almost all bromodeoxyuridine-positive cells in the molecular layer, indicating stimulation of endothelial cell proliferation by cilostazol. These data suggest that cilostazol markedly enhances neovascularization in the hippocampus CA1 area in a mouse model of transient forebrain ischemia, providing a beneficial interface in which both bone marrow-derived endothelial progenitor cells and angioneurins influence neurogenesis in injured tissue. (c) 2010 Wiley-Liss, Inc.

An evidence for adhesion-mediated acquisition of acute myeloid leukemic stem cell-like immaturities

For long-term survival in vitro and in vivo of acute myeloid leukemia cells, their adhesion to bone marrow stromal cells is indispensable. However, it is still unknown if these events are uniquely induced by the leukemic stem cells. Here we show that TF-1 human leukemia cells, once they have formed a cobblestone area by adhering to mouse bone marrow-derived MS-5 cells, can acquire some leukemic stem cell like properties in association with a change in the CD44 isoform-expression pattern and with an increase in a set of related microRNAs. These findings strongly suggest that at least some leukemia cells can acquire leukemic stem cell like properties in an adhesion-mediated stochastic fashion.

Cardiac progenitor-stromal cell interaction: an important player in myocardial development

There is growing support to the notion that adult mammalian heart harbours a pool of resident cardiac stem cells (CSCs). This discovery has sparked new optimism in the field of cardiac regenerative medicine based on the idea that stimulating the myocardial endogenous repair may hold greater promises than reconstituting the post-infarcted heart by transplanted exogenous stem cells. However, before considering endogenous CSCs as a target of therapeutic options in the heart failure, we need to better understand the complex mechanisms controlling heart morphogenesis, paying attention to the cellular and molecular interactions that cardiomyocyte precursors establish with other cells of the stromal compartment. On these basis, in the present study we co-cultured mouse neonatal cardiomyocytes with mouse bone marrow-derived mesenchymal stromal cells (MSCs) to investigate whether these cells could influence cardiomyocyte development in vitro. We found that proliferation of cardiac precursors was dramatically enhanced by direct co-culture with MSC as compared with the single culture system, as judged by time lapse video-microscopy and cyclin A expression/nuclear translocation. We also showed that the proliferative response of the neonatal cardiomyocytes involved the activation of Notch1 receptor by its ligand Jagged 1 and that MSCs sustained this pathway through the constitutive expression of high levels of Jagged 1. This was supported by the observations that cardiomyocytes in contact with MSCs revealed a stronger immunoreactivity for the activated Notchintracellular domain (Notch-ICD) as compared to those in single culture. In conclusion, these findings reveal a previously unrecognized function of MSCs in regulating cardiomyocyte proliferation and development and suggest that stromal-cardiac muscle cell interactions, beside being crucial for correct myocardial morphogenesis, may represent an interesting target for innovative cell therapy strategies in cardiac regeneration.

Apoptosis Signal-Regulating Kinase 1 Regulates Colitis and Colitis-Associated Tumorigenesis by the Innate Immune Responses

Mitogen-activated protein kinase (MAPK) signaling pathways regulate multiple cellular functions and are implicated in the pathogenesis of inflammatory bowel disease and colitis-associated cancer (CAC). Apoptosis signal-regulating kinase 1 (ASK1) is a MAPK kinase kinase; little is known about the role of ASK1 in colonic disease. We assessed the involvement of ASK1 in the development of intestinal inflammation and CAC. Dextran sodium sulfate (DSS) or Citrobacter rodentium was used to induce colitis in wild-type (WT) and ASK1 knock-out (ASK1(-/-)) mice; CAC was induced by azoxymethane injection followed by repeated intake of DSS by the mice. Primary macrophages were isolated from WT and ASK1(-/-) mice and used to investigate the involvement of ASK1 in innate immune responses. Bone marrow chimeric mice were used to study the contribution of myeloid cells to colitis activity. ASK1 deficiency increased susceptibility to colonic inflammation in both models of colitis. In vitro, ASK1(-/-) macrophages were impaired in their ability to kill bacteria and had increased susceptibility to bacterial-induced apoptosis, because p38 was inactivated. Expression of antiapoptotic genes was greatly reduced in ASK1(-/-) macrophages. WT mice given transplants of ASK1(-/-) mouse-derived bone marrow cells developed more severe DSS-induced colitis than mice with WT-derived bone marrow cells. In the CAC model, ASK1(-/-) mice developed more numerous and larger tumors than WT mice through increased colonic inflammation. ASK1 controls the development of intestinal inflammation and CAC through the regulation of innate immunity.

The Microenvironment of Freeze-Injured Mouse Urinary Bladders Enables Successful Tissue Engineering

Mouse bone marrow-derived cells implanted into freeze-injured bladder walls form smooth muscle layers, but not in intact walls. We determined if the microenvironment within injured urinary bladders was supportive of smooth muscle layer development. The urinary bladders of female nude mice were freeze-injured for 30 s. Three days later, the rate of blood flow in the wounded areas and in comparable areas of intact control urinary bladders was observed by charge-coupled device (CCD) video microscopy. Injured and control bladder walls were also analyzed histologically and cytologically. Growth factor mRNA expression was determined by real-time reverse transcription polymerase chain reaction arrays. The injured regions maintained a partial microcirculation in which blood flow velocity was significantly less than in controls. The injured bladder walls had few typical smooth muscle layers, and blood vessels in the walls had reduced smooth muscle content. The loss of smooth muscle cells in the bladder walls may have resulted in the formation of large porous spaces seen by scanning electron microscopy of the injured areas. The expression of nineteen growth-related mRNAs, including secreted phosphoprotein 1, inhibin beta-A, glial cell line-derived neurotrophic factor, and transforming growth factor beta1, were significantly upregulated in the injured urinary bladders. In conclusion, the microenvironment in freeze-injured urinary bladders enables successful tissue engineering.

Ku protein regulates interferon regulatory factor 1 expression in response to CpG-ODN (135.39)

Abstract Interferon regulatory factor 1 (IRF1) expression is induced in response to toll-like receptor 9 (TLR9) stimulation by bacterial and viral CpG-DNA. Recent studies have demonstrated that IRF1's activity is specific to promoting the expression of interleukin-12p70, but not p40, in a novel signaling pathway that is myeloid differentiation protein-88 (MyD88)-dependent yet nuclear factor kappa B (NF-κB)-independent. Further evidence suggests that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), is upstream of IRF1 activation and required for its expression and subsequent production of interferon in response to CpG- oligodeoxynucleotides (ODNs) stimulation. Here we have demonstrated that regulatory protein Ku is critical for IRF1 expression in response to CpG-ODN stimulation and the subsequent production of IL12p70. In Ku70 and Ku80 knock out mice bone marrow-derived dendritic cells (BMDCs) IRF1 expression was nearly abolished in response to CpG-ODN stimulation, and IL12p70 production was reduced to one fourth that of their wild type counterparts, while IL12p40 levels showed no differences. Our results suggest a novel pathway where Ku proteins regulate DNA-PKcs activation in response to CpG stimulation critical for the expression of IRF1 and the subsequent antiviral response. This research is supported by the GAANN Training Grant.

Biocompatibility of Poly-ε-caprolactone-hydroxyapatite composite on mouse bone marrow-derived osteoblasts and endothelial cells

BackgroundTissue-engineered bone may be developed by seeding the cells capable of both osteogenesis and vascularization on biocompatible composite scaffolds. The current study investigated the performance of mice bone marrow-derived osteogenic cells and endothelial cells as seeded on hydroxyapatite (HA) and poly-ε-caprolactone (PCL) composite scaffolds.MethodsMononuclear cells were induced to osteoblasts and endothelial cells respectively, which were defined by the expression of osteocalcin, alkaline phosphatase (ALP), and deposits of calcium-containing crystal for osteoblasts, or by the expression of vascular endothelial growth factor receptor-2 (VEGFR-2) and von Willebrand factor (vWF), and the formation of a capillary network in Matrigel™ for endothelial cells. Both types of cell were seeded respectively on PCL-HA scaffolds at HA to PCL weight ratio of 1:1, 1:4, or 0:1 and were evaluated using scanning electron microscopy, ALP activity (of osteoblasts) and nitric oxide production (of endothelial cells) plus the assessment of cell viability.ResultsThe results indicated that HA led to a positive stimulation of osteoblasts viability and ALP activity, while HA showed less influence on endothelial cells viability. An elevated nitric oxide production of endothelial cells was observed in HA-containing group.ConclusionSupplement of HA into PCL improved biocompatible for bone marrow-derived osteoblasts and endothelial cells. The PCL-HA composite integrating with two types of cells may provide a useful system for tissue-engineered bone grafts with vascularization.

Effective Immunotherapy against Murine Gliomas Using Type 1 Polarizing Dendritic Cells—Significant Roles of CXCL10

In an attempt to develop effective vaccines against central nervous system (CNS) tumors, we evaluated the ability of vaccines with standard dendritic cells (DC) versus type 1 polarizing DCs (DC1) to induce glioma-specific type 1 CTLs with CNS tumor-relevant homing properties and the mechanism of their action. C57BL/6 mouse-derived bone marrow cells were cultured with mouse granulocyte/macrophage colony-stimulating factor (GM-CSF) for 6 days, and CD11c(+) cells were subsequently cultured with GM-CSF, rmIFN-gamma, rmIFN-alpha, rmIL-4, and polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose for 24 hours to generate DC1s. In analogy to their human counterparts, mouse DC1s exhibited surface marker profiles of mature DCs and produced high levels of IL-12 and CXCL10. Importantly for their application as cancer vaccines, such DC1s stably retained their type 1 phenotype even when exposed to type 2-promoting or regulatory T cell (Treg)-promoting environments. Consistently, mouse DC1s induced antigen-specific type 1 CTLs more efficiently than nonpolarized DCs in vitro. DC1s given s.c. migrated into draining lymph nodes, induced antigen-specific CTLs, and suppressed Treg accumulation. In addition, s.c. immunization with DC1s loaded with glioma-associated antigen (GAA)-derived CTL epitope peptides prolonged the survival of CNS GL261 glioma-bearing mice, which was associated with efficient CNS glioma homing of antigen-specific CTLs. Intratumoral injections of GAA peptide-loaded DC1s further enhanced the anti-CNS glioma effects of DC1-based s.c. immunization. Interestingly, the antitumor functions were abrogated with CXCL10(-/-) mouse-derived DC1s. Collectively, these findings show the anti-CNS glioma effects of DC1-based therapy and a novel role of CXCL10 in the immunologic and therapeutic activity of DC-based cancer vaccines.

Abstract 1743: Stem/Progenitor Markers Sca-1 versus C-kit and CD31 Determine Angiogenic Potential of Mouse Bone Marrow-derived Endothelial Progenitor Cells

Background: Endothelial progenitor cells (EPCs) have been shown to have angiogenic potential contributing to neovascularization. However, the definition of EPC, including surface marker expression of EPCs promoting vasculo-/angiogenesis in ischemic tissue, remains uncertain. We hypothesized that stem/progenitor (c-kit vs. sca-1) and endothelial cell (EC) markers (CD31) may identify cells with enhanced EPC potential. Methods and Results: Mononuclear cells (MNCs) were isolated from mouse bones, and Lin+ cells were depleted by magnetic cell sorting. Lin- cells were further sorted with the following markers (% of total MNCs) by FACS: c-kit+ (1.87%), sca-1+ (0.6%), c-kit+ /CD31+ (1.1%) and sca-1+ /CD31+ (0.28%). Non-sorted MNCs were used as a control. To examine EC phenotype in culture, cells were labeled with DiI and co-cultured with mature ECs (human microvascular endothelial cells: HMVECs). The percent incorporation of DiI labeled cells into HMVEC tube structures 12 hours after co-culture and BS1-lectin positivity/acLDL uptake were: sca-1+ /CD31+ cells (87 ± 2%) > c-kit+ /CD31+ (79 ± 8%) > sca-1+ (62 ± 8%) > c-kit+ (59 ± 5%) > MNC (50 ± 3% ) . Next, we examined homing capacity of these cells to ischemic myocardium using a mouse myocardial infarction (MI) model. DiI-labeled cells (5x10 4 , IV) were injected to splenectomized mice 3 days after MI, and the hearts were excised 24 hours after the cell injection for histological analysis. Interestingly, the number of recruited/retained DiI-labeled-cells in the MI hearts exactly replicated the findings of the in vitro tube formation assay (cells/HPF): sca-1+ /CD31+ (108 ± 26) > c-kit+ /CD31+ (77 ± 16) > sca-1+ (71 ± 14) > c-kit+ (67 ± 1) > MNCs (48 ± 6) , suggesting that sca-1+ /CD31+ cells might have great functional activities as endothelial precursors. Conclusions: Both stem/progenitor marker Sca-1 and EC marker CD31 expressing EPCs exhibited high potential angiogenic capacity with EC phenotypic features compared with c-kit expressing cells. Our data suggest that Sca-1+ /CD31+ cells may represent EPC-rich cell population, and Sca-1/CD31 could be useful markers to enrich for cells with EPC potential. Ongoing studies will determine the in vivo characteristics of these cells for ischemic tissue repair.

Implanted Mouse Bone Marrow-Derived Cells Reconstruct Layered Smooth Muscle Structures in Injured Urinary Bladders

This study is a preliminary investigation to determine if bone marrow-derived cells, when implanted into freeze-injured urinary bladders, differentiate into smooth muscle cells and reconstruct smooth muscle layers. Bone marrow cells were harvested from femurs of male ICR mice and cultured in collagen-coated dishes for 7 days. After 5 days of culture, the cells were transfected with green fluorescent protein (GFP) genes for identification in recipient tissues. Three days prior to implantation, the posterior urinary bladder walls of female nude mice were injured with an iron bar refrigerated by dry ice. Seven days after the culture and 3 days after the injury, adherent, proliferating GFP-labeled bone marrow-derived cells (1.0 x 10(5) cells) were implanted into the injured regions. For controls, a cell-free solution was injected. At 14 days after implantation, the experimental urinary bladders were analyzed by histological, gene expression, and cystometric investigations. Just prior to implantation, the injured regions did not have any smooth muscle layers. After 14 days, the implanted cells surviving in the recipient tissues were detected with GFP antibody. The implanted regions had distinct smooth muscle layers composed of regenerated smooth muscle marker-positive cells. The implanted GFP-labeled cells differentiated into smooth muscle cells that formed into layers. The differentiated cells contacted each other within the implanted region as well as smooth muscle cells of the host. As a result, the reconstructed smooth muscle layers were integrated into the host tissues. Control mice injected with cell-free solution developed only few smooth muscle cells and no layers. Cystometric investigations showed that mice with implanted the cells developed bladder contractions similar to normal mice, whereas control mice did not. In summary, mouse bone marrow-derived cells can reconstruct layered smooth muscle structures in injured bladders to remediate urinary dysfunction.

Dcir deficiency causes development of autoimmune diseases in mice due to excess expansion of dendritic cells

The dendritic cell immunoreceptor (official gene symbol Clec4a2, called Dcir here) is a C-type lectin receptor expressed mainly in dendritic cells (DCs) that has a carbohydrate recognition domain in its extracellular portion and an immunoreceptor tyrosine-based inhibitory motif, which transduces negative signals into cells, in its cytoplasmic portion. We found high Dcir expression in the joints of two mouse rheumatoid arthritis models. Because the structural characteristics of Dcir suggest that it may have an immune regulatory role, and because autoimmune-related genes are mapped to the DCIR locus in humans, we generated Dcir-/- mice to learn more about the pathological roles of this molecule. We found that aged Dcir-/- mice spontaneously develop sialadenitis and enthesitis associated with elevated serum autoantibodies. Dcir-/- mice showed a markedly exacerbated response to collagen-induced arthritis. The DC population was expanded excessively in aged and type II collagen-immunized Dcir-/- mice. Upon treatment with granulocyte-macrophage colony-stimulating factor, Dcir-/- mouse-derived bone marrow cells (BMCs) differentiated into DCs more efficiently than did wild-type BMCs, owing to enhanced signal transducer and activator of transcription-5 phosphorylation. These observations indicate that Dcir is a negative regulator of DC expansion and has a crucial role in maintaining the homeostasis of the immune system.

Immunotherapeutic activity of a conjugate of a Toll-like receptor 7 ligand

The immunotherapeutic activity of Toll-like receptor (TLR) activators has been difficult to exploit because of side effects related to the release and systemic dispersion of proinflammatory cytokines. To overcome this barrier, we have synthesized a versatile TLR7 agonist, 4-[6-amino-8-hydroxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzaldehyde (UC-1V150), bearing a free aldehyde that could be coupled to many different auxiliary chemical entities through a linker molecule with a hydrazine or amino group without any loss of activity. UC-1V150 was covalently coupled to mouse serum albumin (MSA) at a 5:1 molar ratio to yield a stable molecule with a characteristically altered UV spectrum. Compared with the unconjugated TLR7 agonist, the UC-1V150/MSA was a 10- to 100-fold more potent inducer of cytokine production in vitro by mouse bone marrow-derived macrophage and human peripheral blood mononuclear cells. When administrated to the lung, the conjugate induced a prolonged local release of cytokines at levels 10-fold or more higher than those found in serum. Under the same conditions, the untethered TLR7 ligand induced quick systemic cytokine release with resultant toxicity. In addition, two pulmonary infectious disease models were investigated wherein mice were pretreated with the conjugate and then challenged with either Bacillus anthracis spores or H1N1 influenza A virus. Significant delay in mortality was observed in both disease models with UC-1V150/MSA-pretreated mice, indicating the potential usefulness of the conjugate as a localized and targeted immunotherapeutic agent.

Anti-tumour potential of a gallic acid-containing phenolic fraction from Oenothera biennis

A phenolic fraction purified form defatted seeds of Oenothera biennis promoted selective apoptosis of human and mouse bone marrow-derived cell lines following first-order kinetics through a caspase-dependent pathway. In non-leukemia tumour cell lines, such as human colon carcinoma CaCo 2 cells and mouse fibrosarcoma WEHI164 cells, this fraction inhibited 3H-thymidine incorporation but not cell death or cell cycle arrest. Human peripheral blood mononuclear cells showed low sensitivity to treatment. Single bolus injection of the phenolic fraction could delay the growth of established myeloma tumours in syngeneic animals. HPLC and mass spectrometry analysis revealed that the fraction contains gallic acid. However, the biological activity of the fraction differs from the activity of this phenol and hence it should be attributed to other co-purified molecules which remain still unidentified.

Endogenous bone-marrow-derived stem cells contribute only a small proportion of regenerated myocardium in the acute infarction model

Background Our recent study showed that granulocyte-colony stimulating factor (G-CSF) promoted bone-marrow cells (BMC) to migrate into the infarcted heart and that they differentiated into cardiomyocytes. However, we still do not know to what degree bone-marrow-derived cardiomyocytes contribute to myocardial regeneration after injury. In this study, we verified the proportional contribution of cells from bone marrow (BM) and from non-bone marrow (n-BM) in regenerating neomyocardium after myocardial infarction. Methods Eight C57BL/6 mice were irradiated (900 cGy), and green fluorescent protein (GFP) mouse-derived BMCs (GFP-BMC, 1 × 10 6 cells) were injected. Four weeks later, the left descending coronary artery was ligated. Recombinant human G-CSF (200 μg/kg/day, 8 days) was injected. At 4 weeks after ligation, hearts were fixed for histology. We calculated the proportions of cardiomyocytes derived from BM and n-BM after taking the chimeric rate into consideration. Results The chimeric rate was 54.6% ± 5.9%. At the infarcted border area, the total cell number was 1000.3 ± 56.5/mm 2, and mobilized BM-derived GFP-BMC was 103.3 ± 13.1/mm 2. After compensation with the chimeric rate, we found BM-derived troponin I-positive cells at 23.9 ± 4.1/mm 2, nestin-positive cells at 12.9 ± 2.6/mm 2, and Ki67-positive cells at 18.3 ± 2.6/mm 2, respectively. We found significant differences in the contribution of troponin I-(6.7% ± 1.7% vs 93.3% ± 1.7%), nestin- (2.4 ± 0.5 vs 97.6 ± 0.5), and Ki67-positive (3.9 ± 1.0 vs 96.1 ± 1.0) cells derived from BM and n-BM. Conclusions Bone marrow was one of the origins of regenerated cardiomyocytes; however, the contribution of cells from BM was very small compared with those of n-BM origin in the infarction model.

Plasticity and Tissue Regenerative Potential of Bone Marrow-Derived Cells

Diverse in vivo studies have suggested that adult stem cells might have the ability to differentiate into cell types other than those of the tissues in which they reside or derive during embryonic development. This idea of stem cell "plasticity" has led investigators to hypothesize that, similar to embryonic stem cells, adult stem cells might have unlimited tissue regenerative potential in vivo, and therefore, broad and novel therapeutic applications. Since the beginning of these observations, our group has critically examined these exciting possibilities for mouse bone marrow-derived cells by taking advantage of well-characterized models of tissue regeneration, Cre/lox technology, and novel stem cell isolation protocols. Our experimental evidence does not support plasticity of hematopoietic stem cells as a frequent physiological event, but rather indicates that cell fusion could account for reported cases of hematopoietic stem cell plasticity or "transdifferentiation" in vivo. Our studies highlight the need for meticulous technical controls during the isolation, transplantation, tracking, and analysis of bone marrow-derived cells during in vivo studies on plasticity. Further studies will be necessary to better define experimental conditions and criteria to unequivocally prove or reject plasticity in vivo. In this review, we focus on results from several studies from our laboratory, and discuss their conclusions and implications.

Mast Cells Acquire Monocyte-Specific Gene Expression and Monocyte-Like Morphology by Overproduction of PU.1

PU.1 is a myeloid- and lymphoid-specific transcription factor that belongs to the Ets family. Recently, we found that overproduction of PU.1 in mouse bone marrow-derived hemopoietic progenitor cells induced monocyte-specific gene expression and caused their monocyte-like morphological change. In the present study, PU.1 was overproduced by using retrovirus expression system in differentiated bone marrow-derived mast cells. By overexpression of PU.1, cell surface expression of MHC class II, CD11b, CD11c, and F4/80 was induced, accompanied by reduced expression of c-kit, a mast cell-specific marker. Morphology of PU.1-transfected cells was altered toward monocyte-like one. PU.1-overproducing cells acquired T cell stimulatory ability and showed an increase in response to LPS stimulation, while response through FcepsilonRI was markedly reduced by overproduction of PU.1. These results suggest that the differentiated mast cells still have potential to display monocytic features. When PU.1 was overproduced in a different type of mast cell, peritoneal mast cells, similar monocyte-like morphological change, and the expression of CD11b and F4/80 were induced. However, surface level of CD11c and MHC class II was not affected. These results indicate that the potential capacity to exhibit monocytic features is different between both the mast cells.

G-CSF Promotes Bone Marrow Cells to Migrate into Infarcted Mice Heart, and Differentiate into Cardiomyocytes

A recent study showed that granulocyte-colony stimulating factor (G-CSF) treatment improved the infarcted cardiac function. Although mobilized stem cells may affect it, the mechanism is unclear. In this study, we investigated the origins of stem cells and phenotypic changes of the migrated cells, and evaluated the efficacy of G-CSF. Eighteen C57BL/6 mice were irradiated (900 cGy) and GFP mouse-derived bone marrow cells (GFP-BMC: 10(6) cells) were injected via a tail vein followed by splenectomy 4 weeks later. Ligation of the left descending coronary artery was performed 2 weeks later. Recombinant human G-CSF (200 microg/kg/day) was injected for 3 days before and 5 days after ligation (group 1, n = 10). Saline was injected in group 2 (n = 8). Four weeks after infarction, hearts and other organs were fixed for histology. The survival rate after postoperative day 3 in group 1 was 100%, while that in group 2 was 50% (p = 0.03). Bone marrow-derived GFP cells (BMD-GFP) in group 1 (103.3+/-71.9/mm2) were located at the infarcted border area significantly more than those in group 2 (43.6+/-23.7/mm2) (p < 0.0001). BMD-GFP cells were positive for troponin I (16.6%), myosin heavy chain-slow (16.7%), and nestin (8.8%) in group 1. Ki-67-positive BMD-GFP in group 1 (10.0+/-7.0/mm2) were significantly more than those in group 2 (4.8+/-6.1/mm2) (p = 0.01). G-CSF increased the survival rate after infarction. G-CSF promoted BMC to migrate into the infarcted border area. Bone marrow was one of the origins of regenerated cardiomyocytes.