Reprogramming of bone marrow mesenchymal stem cells into cardiomyocytes

We have isolated a cardiomyogenic cell line (CMG cell) from murine bone marrow mesenchymal stem cells. The cells showed a fibroblast-like morphology, but the morphology changed after 5-azacytidine exposure. They began spontaneous beating after 2 weeks, and expressed ANP and BNP. Electron microscopy revealed a cardiomyocyte-like ultrastructure. These cells had several types of action potentials: sinus-node-like and ventricular-cell-like action potentials. The isoform of contractile protein genes indicated that their muscle phenotype was similar to fetal ventricular cardiomyocytes. They expressed alpha 1A, alpha 1B, alpha 1D, beta 1, and beta 2 adrenergic and M1 and M2 muscarinic receptors. Stimulation with phenylephrine, isoproterenol and carbachol increased ERK phosphorylation and second messengers. Isoproterenol increased the beating rate, which was blocked with CGP20712A (beta 1-selective blocker). These findings indicated that cell transplantation therapy for the patients with heart failure might possibly be achieved using the regenerated cardiomyocytes from autologous bone marrow cells in the near future.     

Regeneration of cardiomyocytes from bone marrow: Use of mesenchymal stem cell for cardiovascular tissue engineering

We have isolated a cardiomyogenic cell line (CMG cell) from murine bone marrow mesenchymal stem cells. The cells showed a fibroblast-like morphology, but the morphology changed after 5-azacytidine exposure. They began spontaneous beating after 2 weeks, and expressed ANP and BNP. Electron microscopy revealed a cardiomyocyte-like ultrastructure. These cells had several types of action potentials; sinus node-like and ventricular cell-like action potentials. The isoform of contractile protein genes indicated that their muscle phenotype was similar to fetal ventricular cardiomyocytes. They expressed α_1A, α_1B, α_1D, β₁, and β₂ adrenergic and M₁ and M₂ muscarinic receptors. Stimulation with phenylephrine, isoproterenol and carbachol increased ERK phosphorylation and second messengers. Isoproterenol increased the beating rate, which was blocked with CGP20712A (β₁-selective blocker). These findings indicated that cell transplantation therapy for the patients with heart failure might possibly be achieved using the regenerated cardiomyocytes from autologous bone marrow cells in the near future. This revised version was published online in July 2006 with corrections to the Cover Date.     

Fetal bovine serum enables cardiac differentiation of human embryonic stem cells

During development, cardiac commitment within the mesoderm requires endoderm-secreted factors. Differentiation of embryonic stem cells into the three germ layers in vitro recapitulates developmental processes and can be influenced by supplements added to culture medium. Hence, we investigated the effect of fetal bovine serum (FBS) and KnockOut serum replacement (SR) on germ layers specification and cardiac differentiation of H1 human embryonic stem cells (hESC) within embryoid bodies (EB). At the time of EB formation, FBS triggered an increased apoptosis. As assessed by quantitative PCR on 4-, 10-, and 20-day-old EB, FBS promoted a faster down-regulation of pluripotency marker Oct4 and an increased expression of endodermal (Sox17, alpha-fetoprotein, AFP) and mesodermal genes (Brachyury, CSX). While neuronal and hematopoietic differentiation occurred in both supplements, spontaneously beating cardiomyocytes were only observed in FBS. Action potential (AP) morphology of hESC-derived cardiomyocytes indicated that ventricular cells were present only after 2 months of culture. However, quantification of myosin light chain 2 ventricular (mlc2v)-positive areas revealed that mlc2v-expressing cardiomyocytes could be detected already after 2 weeks of differentiation, but not in all beating clusters. In conclusion, FBS enabled cardiac differentiation of hESC, likely in an endodermal-dependent pathway. Among cardiac cells, ventricular cardiomyocytes differentiated over time, but not as the predominant cardiac cell subtype.     

Bone marrow-derived side population cells are capable of functional cardiomyogenic differentiation

It has been reported that bone marrow (BM)-side population (SP) cells, with hematopoietic stem cell activity, can transdifferentiate into cardiomyocytes and contribute to myocardial repair. However, this has been questioned by recent studies showing that hematopoietic stem cells (HSCs) adopt a hematopoietic cell lineage in the ischemic myocardium. The present study was designed to investigate whether BM-SP cells can in fact transdifferentiate into functional cardiomyocytes. Phenotypically, BM-SP cells were 19.59% (+/-)9.00 CD14+, 8.22% (+/-)2.72 CD34+, 92.93% (+/-)2.68 CD44+, 91.86% (+/-)4.07 CD45+, 28.48% (+/-)2.24 c-kit+, 71.09% (+/-)3.67 Sca-1+. Expression of endothelial cell markers (CD31, Flk-1, Tie-2 and VEGF-A) was higher in BM-SP cells than whole BM cells. After five days of co-culture with neonatal cardiomyocytes, 7.2% (+/-)1.2 of the BM-SP cells expressed sarcomeric alpha-actinin as measured by flow cytometry. Moreover, BM-SP cells co-cultured on neonatal cardiomyocytes fixed to inhibit cell fusion also expressed sarcomeric alpha-actinin. The co-cultured BM-SP cells showed neonatal cardiomyocyte-like action potentials of relatively long duration and shallow resting membrane potential. They also generated calcium transients with amplitude and duration similar to those of neonatal cardiomyocytes. These results show that BM-SP cells are capable of functional cardiomyogenic differentiation when co-cultured with neonatal cardiomyocytes.     

Osteoblast-related gene expression of bone marrow cells during the osteoblastic differentiation induced by type I collagen

Bone marrow contains multipotent cells that differentiate into fibroblasts, adipocytes, and osteoblasts. Recently we found that type I collagen matrix induced the osteoblastic differentiation of bone marrow cells. Three weeks after cells were cultured with type I collagen, they formed mineralized tissues. In this study, we investigated the expression of osteoblast-related genes (alkaline phosphatase, osteocalcin, bone sialoprotein, osteopontin, and cbfa-1) during the osteoblastic differentiation. The expression of alkaline phosphatase and osteopontin genes increased time-dependently during the osteoblastic differentiation. Osteocalcin and bone sialoprotein genes were expressed in cells that formed mineralized tissues, and both were expressed only after cells reached the mineralized tissue-formation stage. On the other hand, the cbfa-1 gene was expressed from the early differentiation stage. The Asp-Gly-Glu-Ala (DGEA) amino acid domain of type I collagen interacts with the alpha2beta1 integrin receptor on the cell membrane and mediates extracellular signals into cells. When the collagen-integrin interaction was interrupted by the addition of DGEA peptide to the culture, the expression of osteoblastic phenotypes of bone marrow cells was inhibited. These findings imply that the collagen-alpha2beta1 integrin interaction is an important signal for the osteoblastic differentiation of bone marrow cells.     

'Working' cardiomyocytes exhibiting plateau action potentials from human placenta-derived extraembryonic mesodermal cells

The clinical application of cell transplantation for severe heart failure is a promising strategy to improve impaired cardiac function. Recently, an array of cell types, including bone marrow cells, endothelial progenitors, mesenchymal stem cells, resident cardiac stem cells, and embryonic stem cells, have become important candidates for cell sources for cardiac repair. In the present study, we focused on the placenta as a cell source. Cells from the chorionic plate in the fetal portion of the human placenta were obtained after delivery by the primary culture method, and the cells generated in this study had the Y sex chromosome, indicating that the cells were derived from the fetus. The cells potentially expressed 'working' cardiomyocyte-specific genes such as cardiac myosin heavy chain 7beta, atrial myosin light chain, cardiac alpha-actin by gene chip analysis, and Csx/Nkx2.5, GATA4 by RT-PCR, cardiac troponin-I and connexin 43 by immunohistochemistry. These cells were able to differentiate into cardiomyocytes. Cardiac troponin-I and connexin 43 displayed a discontinuous pattern of localization at intercellular contact sites after cardiomyogenic differentiation, suggesting that the chorionic mesoderm contained a large number of cells with cardiomyogenic potential. The cells began spontaneously beating 3 days after co-cultivation with murine fetal cardiomyocytes and the frequency of beating cells reached a maximum on day 10. The contraction of the cardiomyocytes was rhythmical and synchronous, suggesting the presence of electrical communication between the cells. Placenta-derived human fetal cells may be useful for patients who cannot supply bone marrow cells but want to receive stem cell-based cardiac therapy.     

Co-culture with cardiomyocytes enhanced the myogenic conversion of mesenchymal stromal cells in a dose-dependent manner

To increase the accessibility of myogenic cells for cell therapy in the infarcted heart, we identified conditions to improve the reproducible conversion of bone marrow mesenchymal stromal cells (BMSCs) into myogenic cells. Such cells may permit functional regeneration following a myocardial infarction. BMSCs derived from green fluorescent protein (GFP) transgenic rats were co-cultured with neonatal rat cardiomyocytes (1:1, 1:10, 1:20, and 1:40 ratios) for 7 days. Some BMSCs contracted synchronously with the neonatal cardiomyocytes, and exhibited action potentials that were confirmed with current clamp recordings. The myogenic phenotype of the BMSCs was confirmed by immunohistochemical staining and flow cytometry (antibodies against cardiac specific α-sarcomeric actinin, Troponin I, MEF-2C). An increase in the number of BMSCs expressing cardiac markers correlated with increasing numbers of neonatal cardiomyocytes in the culture. When BMSCs were co-cultured with DiI-labeled neonatal cardiomyocytes, a small percentage of GFP/DiI/Troponin I triple-positive cells were observed after 7 days. This type of myogenic conversion increased nearly twofold when BMSCs were co-cultured with apoptotic (TNF-α-treated) cardiomyocytes. BMSCs co-cultured with cardiomyocytes acquired a functional myogenic phenotype in a dose-dependent manner. Myogenic conversion increased when the BMSCs were cultured with apoptotic cells.     

Colony-stimulating factor-1 modulates alpha 2-macroglobulin receptor expression in murine bone marrow macrophages.

Primary cultures of murine bone marrow macrophages (BMMs) were prepared from marrow cell suspensions. These cells expressed specific receptors that recognized the transformed conformation of human alpha 2-macroglobulin (alpha 2M) generated by reaction with CH3NH2. alpha 2M receptor expression was regulated by colony-stimulating factor-1 (CSF-1). The BMMs were deprived of CSF-1 for 6 h and then treated with different concentrations of the purified cytokine. After 18 h, binding of 125I-alpha 2M-CH3NH2 was examined at 4 degrees C. Analysis of the saturation isotherms and Scatchard transformations indicated that the KD was not affected by CSF-1 (1.9-2.4 nM), whereas the maximum specific radioligand binding capacity (Bmax) was increased from 5.6 x 10(4) receptors/cell in the absence of CSF-1 to 2.2 x 10(5) and 2.6 x 10(5) receptors/cell for BMMs treated with 1,000 and 10,000 units/ml CSF-1, respectively. The difference in total cellular protein after exposure to different levels of CSF-1 for 18 h was small (1.50-1.92 ng/cell) and not statistically significant. A 6-12-h lag phase was identified between the time of CSF-1 exposure and increased alpha 2M receptor expression. Cycloheximide completely blocked the increase in alpha 2M receptor expression when added simultaneously with the CSF-1; greater than 50% inhibition was observed when the cycloheximide was added up to 8 h later. The RNA synthesis inhibitors, actinomycin D and daunomycin, prevented increased alpha 2M receptor expression when added up to 4 h after the CSF-1, but had no effect at 8 h. At 37 degrees C, uptake and digestion of 125I-alpha 2M-CH3NH2 was increased in BMMs treated with 1,000 units/ml CSF-1 for 18 h compared with untreated cells. These studies demonstrate that CSF-1 increases the expression of alpha 2M receptors in BMMs through a pathway that requires new RNA and protein synthesis. We hypothesize that increased alpha 2M receptor expression may play an important role in cellular growth and differentiation.     

Unusual T cell populations in adult murine bone marrow. Prevalence of CD3+CD4-CD8- and alpha beta TCR+NK1.1+ cells

The T cell populations present in normal murine bone marrow have not been previously analyzed in detail, mainly because of their relative rarity. In order to permit such analyses, bone marrow T cells were enriched by depleting Mac1-positive cells, which constitute 65 to 90% of bone marrow cells (BMC), and then studied by two-color flow cytometry. Analysis of the remaining cells revealed that the T cell profile of adult murine bone marrow is markedly different from that of other lymphoid organs. A very high proportion of bone marrow CD3+ cells (approximately one-third) are CD4-CD8-. CD3+CD4-CD8- cells are much more concentrated among BMC T cells than among thymocytes or splenic T cells, suggesting that bone marrow may be either a site of extrathymic TCR gene rearrangement, or a major site to which such cells home from the thymus. The expression of NK1.1 was also evaluated on Mac1-depleted BMC populations. Surprisingly, up to 39% of alpha beta TCR+ BMC were found to express NK1.1. Most alpha beta TCR+NK1.1+ BMC also expressed CD4 or CD8. NK1.1+ alpha beta TCR+ cells represented a much greater proportion of BMC T cells than of other lymphoid (splenocyte or thymocyte) T cell populations. Mac1-depleted BMC of nude mice contained very few cells with this phenotype. These results are consistent with the hypothesis that NK1.1+ alpha beta TCR+ cells are generated primarily in the thymus of normal animals and migrate preferentially to bone marrow, where they may function as regulatory elements in hematopoiesis.     

Characterization of mice lacking the tetraspanin superfamily member CD151

The tetraspanin membrane protein CD151 is a broadly expressed molecule noted for its strong molecular associations with integrins, especially alpha3beta1, alpha6beta1, alpha7beta1, and alpha6beta4. In vitro functional studies have pointed to a role for CD151 in cell-cell adhesion, cell migration, platelet aggregation, and angiogenesis. It has also been implicated in epithelial tumor progression and metastasis. Here we describe the generation and initial characterization of CD151-null mice. The mice are viable, healthy, and fertile and show normal Mendelian inheritance. They have essentially normal blood and bone marrow cell counts and grossly normal tissue morphology, including hemidesmosomes in skin, and expression of alpha3 and alpha6 integrins. However, the CD151-null mice do show phenotypes in several different tissue types. An absence of CD151 leads to a minor abnormality in hemostasis, with CD151-null mice showing longer average bleeding times, greater average blood loss, and an increased incidence of rebleeding occurrences. CD151-null keratinocytes migrate poorly in skin explant cultures. Finally, CD151-null T lymphocytes are hyperproliferative in response to in vitro mitogenic stimulation.     

Inhibitory effect of NPY on isoprenaline-induced osteoclastogenesis in mouse bone marrow cells

The effect of neuropeptide Y (NPY), a co-transmitter with noradrenaline in peripheral sympathetic nerve fibers, on the osteoclastogenesis in mouse bone marrow cell cultures treated with isoprenaline, a beta-adrenergic receptor (beta-AR) agonist, was examined. The mouse bone marrow cells constitutively expressed mRNAs for the NPY-Y1 receptor and beta2-AR. NPY inhibited the formation of osteoclast-like cells induced by isoprenaline but not that by 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) or soluble receptor activator of nuclear factor-kappaB ligand (RANKL); and it suppressed the production of RANKL and cyclic AMP (cAMP) increased by isoprenaline but not those increased by 1alpha,25(OH)2D3. NPY also inhibited osteoclastogenesis induced by forskolin, an activator of adenylate cyclase; however, it did not inhibit that induced by exogenously supplied dibutyryl cAMP, a cell-permeable cAMP analog that activates cAMP-dependent protein kinase. These results demonstrate that NPY inhibited the isoprenaline-induced osteoclastogenesis by blocking the agonist-elicited increases in the production of cAMP and RANKL in mouse bone marrow cells, suggesting an interaction between NPY and beta-AR agonist in bone resorption.     

Influence of sympathetic innervation on the membrane electrical properties of neonatal rat cardiomyocytes in culture

Co-cultures of rat ventricular myocytes and sympathetic neurons were established. Superior cervical ganglia and ventricles from newborn rats were enzymatically dissociated and plated in a culture dish. Experiments were done between the 3rd (when evidence of neuron-myocyte proximity arises) and the 5th day in culture (before the myocytes become confluent). Simultaneous intracellular recording from a cardiomyocyte and an attached neuron was done using conventional microelectrode techniques (resistance of 60-100 Mohm). The myocytes in co-culture were either quiescent or spontaneously contracting. The contracting cells were either latent pacemaker or ventricular-like myocytes. The action potential (AP) characteristics of cardiomyocytes in co-cultures were comparable to those recorded in cardiomyocytes in pure cultures. Sympathetic innervation of the cardiomyocytes in co-cultures was evidenced by stimulating the neuron and observing an increase in rate of beating in latent pacemaker myocytes (average increase of 19.4 +/- 4.6%). In quiescent cardiomyocytes, neural stimulation evoked a slow depolarization that can reach threshold and initiate APs in the cell. This response is similar to slow excitatory postsynaptic potentials (EPSPs) observed in other synapses. Slow ESPSs could also be recorded in spontaneous beating cells, made quiescent by nifedipine (1x10(-6)-1x10(-7) M). These results indicate that functional synaptic contacts are developed in co-culture of sympathetic neurons and cardiac myocytes, and slow EPSPs can be evoked in cardiomyocytes as well as in other excitable cells. The sympathetic innervation occurring in culture did not significantly modify the spontaneous AP characteristics of the cardiomyocytes.     

A dual role of the GTPase Rac in cardiac differentiation of stem cells

The function of the GTPase Rac1, a molecular switch transducing intracellular signals from growth factors, in differentiation of a specific cell type during early embryogenesis has not been investigated. To address the question, we used embryonic stem (ES) cells differentiated into cardiomyocytes, a model that faithfully recapitulates early stages of cardiogenesis. Overexpression in ES cells of a constitutively active Rac (RacV12) but not of an active mutant (RacL61D38), which does not activate the NADPH oxydase generating ROS, prevented MEF2C expression and severely compromised cardiac cell differentiation. This resulted in poor expression of ventricular myosin light chain 2 (MLC2v) and its lack of insertion into sarcomeres. Thus ES-derived cardiomyocytes featured impaired myofibrillogenesis and contractility. Overexpression of MEF2C or addition of catalase in the culture medium rescued the phenotype of racV12 cells. In contrast, RacV12 specifically expressed in ES-derived ventricular cells improved the propensity of cardioblasts to differentiate into beating cardiomyocytes. This was attributed to both a facilitation of myofibrillogenesis and a prolongation in their proliferation. The dominant negative mutant RacN17 early or lately expressed in ES-derived cells prevented myofibrillogenesis and in turn beating of cardiomyocytes. We thus suggest a stage-dependent function of the GTPase during early embryogenesis.     

Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with cyclosporin-A

Induced pluripotent stem cells (iPSCs) are novel stem cells derived from adult mouse and human tissues by reprogramming. Elucidation of mechanisms and exploration of efficient methods for their differentiation to functional cardiomyocytes are essential for developing cardiac cell models and future regenerative therapies. We previously established a novel mouse embryonic stem cell (ESC) and iPSC differentiation system in which cardiovascular cells can be systematically induced from Flk1(+) common progenitor cells, and identified highly cardiogenic progenitors as Flk1(+)/CXCR4(+)/VE-cadherin(-) (FCV) cells. We have also reported that cyclosporin-A (CSA) drastically increases FCV progenitor and cardiomyocyte induction from mouse ESCs. Here, we combined these technologies and extended them to mouse and human iPSCs. Co-culture of purified mouse iPSC-derived Flk1(+) cells with OP9 stroma cells induced cardiomyocyte differentiation whilst addition of CSA to Flk1(+) cells dramatically increased both cardiomyocyte and FCV progenitor cell differentiation. Spontaneously beating colonies were obtained from human iPSCs by co-culture with END-2 visceral endoderm-like cells. Appearance of beating colonies from human iPSCs was increased approximately 4.3 times by addition of CSA at mesoderm stage. CSA-expanded human iPSC-derived cardiomyocytes showed various cardiac marker expressions, synchronized calcium transients, cardiomyocyte-like action potentials, pharmacological reactions, and ultra-structural features as cardiomyocytes. These results provide a technological basis to obtain functional cardiomyocytes from iPSCs.     

骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖的促进作用

本文通过制备小鼠骨髓内皮细胞无血清条件培养液(serum-free murine bone marrow endothelial cell conditioned medium, mBMEC-CM),经超滤分为分子量>10 kDa组分和<10 kDa组分,分别观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞集落生成的影响。用Wright’S Giemsa染色计数内皮细胞集落及检测骨髓内皮细胞的vWF,通过[3H]- TdR掺入量,观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞增殖的影响,并用分子杂交方法检测内皮细胞表达的细胞因子,从几个方面来研究mBMEC-CM对骨髓内皮细胞增殖的作用。结果显示,骨髓内皮细胞vWF 检测阳性。mBMEC-CM原液及其分子量>10 kDa组分能刺激骨髓内皮细胞集落增殖,且能明显增加骨髓内皮细胞[3H]-TdR 掺入量;分子量<10 kDa组分对骨髓内皮细胞集落增殖无明显刺激作用,也不能增加骨髓内皮细胞[3H]-TdR掺入量。外源加入IL-6、IL-11、SCF、GM-CSF、VEGF、bFGF 6种细胞因子能明显刺激骨髓内皮细胞集落增殖,SCF、VEGF、bFGF能明显增加骨髓内皮细胞[3H]-TdR掺入量。Atlas array膜杂交实验显示骨髓内皮细胞内源性表达GM-CSF、SCF、MSP-1、endothelin-2、thymosin β10、connective tissue GF、PDGF-A chain、MIP-2α、PlGF、neutrophil activating protein ENA-78、INF-γ、IL-1、IL-6、IL-13、IL-11、inhibin-α等细胞因子的mRNA。上述结果提示,骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖具有促进作用。     

Integrin alpha4beta1 involvement in stromal cell-derived factor-1alpha-promoted myeloma cell transendothelial migration and adhesion: role of cAMP and the actin cytoskeleton in adhesion

The chemokine stromal cell-derived factor-1alpha (SDF-1alpha) is expressed by bone marrow (BM) stromal cells and plays key roles in cell homing to and retention into the bone marrow. In multiple myeloma, blood-borne malignant plasma cells home to the BM and accumulate in contact with stromal cells, implicating myeloma cell migration across endothelium. Myeloma cells express the SDF-1alpha receptor CXCR4, as well as the integrin alpha4beta1, which mediates their attachment to BM stroma. We show here that SDF-1alpha promotes transendothelial migration of purified BM myeloma cells and myeloma-derived NCI-H929 cells, involving a transient upregulation of alpha4beta1-dependent cell adhesion to the endothelium. Characterization of intracellular signaling pathways involved in the modulation by SDF-1alpha of alpha4beta1-mediated myeloma cell adhesion revealed that intracellular cAMP amounts associated with the activation of protein kinase A play key roles in this modulation. Furthermore, a functional link between cAMP actions on the dynamics of actin cytoskeleton, RhoA activation, and alpha4beta1-dependent cell adhesion in response to SDF-1alpha has been found. The regulation of alpha4beta1-mediated myeloma cell adhesion by SDF-1alpha could play key roles during myeloma cell homing into and trafficking inside the BM, and characterization of the molecular events involved in SDF-1alpha-activated modulation of this adhesion will contribute to a better understanding of mechanisms participating in cell migration.     

Role of MAdCAM-1 and its ligand on the homing of transplanted hematopoietic cells in irradiated mice

We examined the expression of VCAM-1 and MAdCAM-1 after bone marrow transplantation (BMT). We also examined the influence of alpha(4)beta(7) integrin blockade on the homing of cells to the bone marrow and spleen. The expression of VCAM-1 and MAdCAM-1 by endothelial cells in the spleen and bone marrow was examined by immunoelectron microscopy using colloidal gold and was analyzed semiquantitatively. To examine the role of alpha(4)beta(7) integrin in donor cells, a homing assay was conducted following alpha(4)beta(7) integrin blockade in bone marrow-derived hematopoietic cells or spleen colony cells. Immediately after BMT, the expression of VCAM-1 and MAdCAM1 markedly decreased, but expression recovered significantly between 12 and 24 h after BMT. VCAM-1 recovered more acutely than MAdCAM-1 from 12 h onward. In the group transplanted with anti-alpha(4)beta(7) integrin antibody-treated bone marrow cells, the numbers of homing cells in the spleen and bone marrow were significantly decreased in an antibody dose-dependent manner. However, the number of homing cells was not different in either the spleen or bone marrow between anti-alpha(4)beta(7) integrin antibody treated and untreated spleen colony cells. It has been reported that alpha(4)beta(1) integrin and its receptor VCAM-1 play major roles in the homing of hematopoietic cells to bone marrow. Our study indicates the importance of MAdCAM-1 and its ligand, alpha(4)beta(7) integrin, in the homing of bone marrow-derived hematopoietic cells, but not spleen colony-derived cells, to both the spleen and bone marrow.     

Type I collagen-induced osteoblastic differentiation of bone-marrow cells mediated by collagen-alpha2beta1 integrin interaction

Bone marrow cells are multipotent cells. When bone marrow cells were cultured with type I collagen matrix gels, they showed high alkaline phosphatase activity, collagen synthesis, and formed mineralized tissues. Furthermore, cells expressed osteocalcin and bone sialoprotein genes, which are osteoblast-specific genes. These findings indicate that type I collagen matrix gels induce osteoblastic differentiation of bone marrow cells. Type I collagen interacts with the alpha 2 beta 1 integrin receptor on the cell membrane and mediates extracellular signals into cells. DGEA peptide is a cell-binding domain of type I collagen molecule. When collagen-integrin interaction was interrupted by the addition of Asp-Gly-Glu-Ala (DGEA) peptide to the culture, the expression of osteoblastic phenotypes of bone marrow cells was inhibited. Furthermore, anti-alpha 2 integrin antibody, which interacts with alpha subunit of integrin and blocks the binding of integrin with collagen, suppressed the expression of osteoblastic phenotypes. These findings imply that collagen-alpha 2 beta 1 integrin interaction is an important signal for the osteoblastic differentiation of bone marrow cells.     

Macrophage inflammatory protein (MIP)-1 beta abrogates the capacity of MIP-1 alpha to suppress myeloid progenitor cell growth

The effects of recombinant murine macrophage inflammatory protein (MIP)-1 beta and MIP-2 on the suppressive activity of MIP-1 alpha were tested using colony formation by human and murine bone marrow burst-forming unit-erythroid (BFU-E), colony-forming unit-granulocyte erythroid macrophage, megakaryocyte (CFU-GEMM), and colony-forming unit-granulocyte macrophage (CFU-GM) progenitor cells. MIP-1 beta, but not MIP-2, when added with MIP-1 alpha to cells, blocked the suppressive effects of MIP-1 alpha on both human and murine BFU-E, CFU-GEMM, and CFU-GM colony formation. Similar results were observed regardless of the early acting cytokines used: human rGM-CSF plus human rIL-3, and two recently described potent cytokines, a genetically engineered human rGM-CSF/IL-3 fusion protein and MGF, a c-kit ligand. The more potent the stimuli, the greater the suppressive activity noted. Pulse treatment of hu bone marrow cells with MIP-1 alpha at 4 degrees C for 1 h was as effective in inhibiting colony formation as continuous exposure of cells to MIP-1 alpha, and the pulsing effect with MIP-1 alpha could not be overcome by subsequent exposure of cells to MIP-1 beta. Also, pulse exposure of cells to MIP-1 beta blocked the activity of subsequently added MIP-1 alpha. For specificity, the action of a nonrelated myelosuppressive factor H-ferritin, was compared. MIP-1 alpha and H-ferritin were shown to act on similar target populations of early BFU-E, CFU-GEMM, and CFU-GM. MIP-1 beta did not block the suppressive activity of H-ferritin. Also, hemin and an inactive recombinant human H-ferritin mutein counteracted the suppressive effects of the wildtype H-ferritin molecule, but did not block the suppressive effects of MIP-1 alpha. These results show that MIP-1 beta's ability to block the action of MIP-1 alpha is specific. In addition, the results suggest that MIP-1 alpha and MIP-beta can, through rapid action, modulate early myeloid progenitor cell proliferation.