In vivo and in vitro differentiation of myocytes from human bone marrow-derived multipotent progenitor cells

OBJECTIVE: Recent studies have shown that bone marrow (BM) contains cells capable of differentiating into myocytes in vivo. However, addition of demethylation drugs has been necessary to induce myocyte differentiation from BM cells in vitro, and precise mechanisms of BM cells' conversion to myocytes and the origin of those cells have not been established. We investigated the expression of myogenic markers during differentiation and maturation of myocytes from BM-derived multipotent adult progenitor cells (MAPC) under physiological culture condition. MATERIALS AND METHODS: Frozen BM samples from 21 healthy donors were used as a source of MAPC. To induce myocyte differentiation MAPC was cultured in the presence of 5% FCS, VEGF, bFGF, and IGF-1, and the expressions of myocyte markers were examined at various time points. We also investigated engraftment and differentiation of MAPC-derived myocytes in vivo. RESULTS: Frozen BM-derived MAPC, cultured under the physiological myogenic condition, demonstrated spatial expression patterns of several myocyte markers similar to that of authentic myocyte differentiation. When injected into murine muscles, MAPC treated with the myogenic condition engrafted and differentiated into myocyte marker-positive cells and myotubes in vivo. CONCLUSION: For the first time, we were able to induce myocyte formation from BM cells under the physiological condition in vitro and demonstrated that treating cells with this condition prior to intramuscular injection increased efficiency of engraftment and differentiation in vivo.     

Evidence for nonclonal hematopoietic progenitor cell populations in bone marrow of patients with myelodysplastic syndromes

Clonality of marrow hematopoietic progenitor cells in myelodysplastic syndromes (MDS) was analyzed by X-chromosome inactivation pattern using polymerase chain reaction (PCR). Five female patients were included in this study; two with refractory anemia (RA) and three with RA with excess blasts (RAEB). They were heterozygous for BstXI restriction fragment length polymorphisms (RFLP) of the X-chromosome-linked phosphoglycerate kinase (PGK) gene. In each patient, erythroid and nonerythroid colonies, grown in the presence of erythropoietin and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibited no remarkable difference in clonal constitution. Two patients showed only one methylation pattern, suggesting the monoclonal origin of hematopoietic progenitor cells. Colonies of two other patients exhibited predominant and minor methylation patterns in PGK gene, indicating that nonclonal progenitor cells remain a minor population. The bone marrow of one patient appeared to contain a greater proportion of nonclonal progenitors. Stem cell factor (SCF), a potent colony- stimulating factor, enhanced both erythroid and nonerythroid colony formation. However, it did not notably alter the clonal constitutions. We conclude that nonclonal hematopoietic progenitor cells can persist in a substantial number of MDS patients.     

The small population of PIG-A mutant cells in myelodysplastic syndromes do not arise from multipotent hematopoietic stem cells

Background

Patients with paroxysmal nocturnal hemoglobinuria harbor clonal glycosylphosphatidylinositol-anchor deficient cells arising from a multipotent hematopoietic stem cell acquiring a PIG-A mutation. Many patients with aplastic anemia and myelodysplastic syndromes also harbor small populations of glycosylphosphatidylinositol-anchor deficient cells. Patients with aplastic anemia often evolve into paroxysmal nocturnal hemoglobinuria; however, myelodysplastic syndromes seldom evolve into paroxysmal nocturnal hemoglobinuria. Here, we investigate the origin and clonality of small glycosylphosphatidylinositol-anchor deficient cell populations in aplastic anemia and myelodysplastic syndromes.

Design and Methods

We used peripheral blood flow cytometry to identify glycosylphosphatidylinositol-anchor deficient blood cells, a proaerolysin-resistant colony forming cell assay to select glycosylphosphatidylinositol-anchor deficient progenitor cells, a novel T-lymphocyte enrichment culture assay with proaerolysin selection to expand glycosylphosphatidylinositol-anchor deficient T lymphocytes, and PIG-A gene sequencing assays to identify and analyze PIG-A mutations in patients with aplastic anemia and myelodysplastic syndromes.

Results

Twelve of 15 aplastic anemia patients were found to harbor a small population of glycosylphosphatidylinositol-anchor deficient granulocytes; 11 of them were found to harbor a small population of glycosylphosphatidylinositol-anchor deficient erythrocytes, 10 patients were detected to harbor glycosylphosphatidylinositol-anchor deficient T lymphocytes, and 3 of them were detected only after T-lymphocyte enrichment in proaerolysin selection. PIG-A mutation analyses on 3 patients showed that all of them harbored a matching PIG-A mutation between CFU-GM and enriched T lymphocytes. Two of 26 myelodysplastic syndromes were found to harbor small populations of glycosylphosphatidylinositol-anchor deficient granulocytes and erythrocytes transiently. Bone marrow derived CD34⁺ cells from 4 patients grew proaerolysin-resistant colony forming cells bearing PIG-A mutations. No glycosylphosphatidylinositol-anchor deficient T lymphocytes were detected in myelodysplastic syndrome patients.

Conclusions

In contrast to aplastic anemia and paroxysmal nocturnal hemoglobinuria, where PIG-A mutations arise from multipotent hematopoietic stem cells, glycosylphosphatidylinositol-anchor deficient cells in myelodysplastic syndromes appear to arise from more committed progenitors.     

In vitro and in vivo effects of recombinant interferon gamma on the growth of hematopoietic progenitor cells from patients with myelodysplastic syndrome

Recombinant interferon gamma (rIFN-gamma) has been shown to have antiproliferative effects on normal and leukemic hematopoietic cells, to induce cell differentiation and to modulate hematopoietic growth factor production. We have studied the effects of rIFN-gamma on the growth of hematopoietic progenitors from 3 patients with myelodysplastic syndrome who were treated with rIFN-gamma (0.01 mg/m2 given subcutaneously three times a week) as part of an Italian pilot study. When bone marrow cells were cultured in semisolid medium in the continuous presence of rIFN-gamma (10-10(4) U/ml), inhibition of colony formation was the most common response. However, an enhancement of hematopoietic progenitor growth was observed in one patient at the lowest concentration tested (10 U/ml). Preincubation of bone marrow mononuclear cells with low concentrations of rIFN-gamma in suspension culture for 5 days induced or enhanced in vitro colony formation in two cases; again, higher concentrations resulted in inhibition of hematopoietic progenitor growth. Two patients showed a slight improvement of in vitro progenitor growth after one month of treatment with rIFN-gamma. Although preliminary, these data indicate that rIFN-gamma may have both stimulatory and inhibitory effects on myelodysplastic hematopoiesis, depending on both the effective concentrations and the interactions with accessory cells.     

In vitro and in vivo arterial differentiation of human multipotent adult progenitor cells

Many stem cell types have been shown to differentiate into endothelial cells (ECs); however, their specification to arterial or venous endothelium remains unexplored. We tested whether a specific arterial or venous EC fate could be induced in human multipotent adult progenitor cells (hMAPCs) and AC133(+) cells (hAC133(+)). In vitro, in the presence of VEGF(165), hAC133(+) cells only adopted a venous and microvascular EC phenotype, while hMAPCs differentiated into both arterial and venous ECs, possibly because hMAPCs expressed significantly more sonic hedgehog (Shh) and its receptors as well as Notch 1 and 3 receptors and some of their ligands. Accordingly, blocking either of those pathways attenuated in vitro arterial EC differentiation from hMAPCs. Complementarily, stimulating these pathways by addition of Delta-like 4 (Dll-4), a Notch ligand, and Shh to VEGF(165) further boosted arterial differentiation in hMAPCs both in vitro and in an in vivo Matrigel model. These results represent the first demonstration of adult stem cells with the potential to be differentiated into different types of ECs in vitro and in vivo and provide a useful human model to study arteriovenous specification.     

In vitro biology of human hematopoietic stem and progenitor cells

Hematopoietic stem and progenitor cells from human umbilical cord blood have been the focus of both basic and clinical research during the last 20 years. It has been clearly demonstrated that such sells possess higher proliferation and expansion potentials, as compared to their adult counterparts, and their capacity to reconstitute the hematopoietic system of mammals has also been shown. Different In vitro systems have been used to characterize the biology of these hematopoietic cells and some culture methods are being currently used to expand the numbers of such cells for clinical purposes.     

Multiple myeloma-related deregulation of bone marrow-derived CD34+ hematopoietic stem and progenitor cells

Multiple myeloma (MM) is a clonal plasma cell disorder frequently accompanied by hematopoietic impairment. We show that hematopoietic stem and progenitor cells (HSPCs), in particular megakaryocyte-erythrocyte progenitors, are diminished in the BM of MM patients. Genomic profiling of HSPC subsets revealed deregulations of signaling cascades, most notably TGFβ signaling, and pathways involved in cytoskeletal organization, migration, adhesion, and cell-cycle regulation in the patients. Functionally, proliferation, colony formation, and long-term self-renewal were impaired as a consequence of activated TGFβ signaling. In accordance, TGFβ levels in the BM extracellular fluid were elevated and mesenchymal stromal cells (MSCs) had a reduced capacity to support long-term hematopoiesis of HSPCs that completely recovered on blockade of TGFβ signaling. Furthermore, we found defective actin assembly and down-regulation of the adhesion receptor CD44 in MM HSPCs functionally reflected by impaired migration and adhesion. Still, transplantation into myeloma-free NOG mice revealed even enhanced engraftment and normal differentiation capacities of MM HSPCs, which underlines that functional impairment of HSPCs depends on MM-related microenvironmental cues and is reversible. Taken together, these data implicate that hematopoietic suppression in MM emerges from the HSPCs as a result of MM-related microenvironmental alterations.     

Deficiency of pluripotent hemopoietic progenitor cells in myelodysplastic syndromes

Summary Pluripotent (CFU-MIX), erythroid (BFU-E) and granulocyte/macrophage (CFU-GM) progenitor cells were examined in bone marrow (BM) from 23 patients with myelodysplastic syndromes (MDS). Patients were grouped according to the FAB classification: Refractory anemia (RA), n=3; RA with ring sideroblasts (RARS), n=3; RA with excess of blasts (RAEB), n=8; RA with excess of blasts in transformation (RAEBt), n=7; chronic myelomonocytic leukemia (CMML), n=2. In FAB groups RA, RARS, RAEB and RAEBt CFU-GM concentrations were normal or decreased but both CMML-patients had increased CFU-GM values. Abnormal cluster growth was observed in 9 of 23 MDS-patients. BFU-E colony formation was subnormal in all cases. Mixed-colony assay values were at the lower limit of controls in one patient and decreased in the remaining 22 MDS-patients. A similar growth pattern of hemopoietic progenitor cells was observed in 19 patients with acute nonlymphocytic leukemia (ANLL), who were studied for comparison. These data suggest a quantitative or qualitative/functional defect of the pluripotent progenitor cell compartment as the major cause for the cytopenia in MDS-patients.     

DNA methylation profiling of myelodysplastic syndrome hematopoietic progenitor cells during in vitro lineage-specific differentiation

Deregulated epigenetic mechanisms are likely involved in the pathogenesis of myelodysplastic syndromes (MDSs). Which genes are silenced by aberrant promotor methylation during MDS hematopoiesis has not been equivalently investigated. Using an in vitro differentiation model of human hematopoiesis, we generated defined differentiation stages (day 0, day 4, day 7, day 11) of erythro-, thrombo- and granulopoiesis from 13 MDS patients and seven healthy donors. Promotor methylation analysis of key regulatory genes involved in cell cycle control (p14, p15, p16, CHK2), DNA repair (hMLH1), apoptosis (p73, survivin, DAPK), and differentiation (RARb, WT1) was performed by methylation-specific polymerase chain reaction. Corresponding gene expression was analyzed by microarray (Affymetrix, HG-U133A). We provide evidence that p16, survivin, CHK2, and WT1 are affected by promotor hypermethylation in MDSs displaying a selective International Prognostic Scoring System risk association. A methylation-associated mRNA downregulation for specific hematopoietic lineages and differentiation stages is demonstrated for survivin, CHK2, and WT1. We identified a suppressed survivin mRNA expression in methylated samples during erythropoiesis, whereas WT1 and CHK2 methylation-related reduction of mRNA expression was found during granulopoiesis in all MDS risk types. Our data suggest that lineage-specific methylation-associated gene silencing of survivin, CHK2, and WT1 in MDS hematopoietic precursor cells may contribute to the MDS-specific phenotype     

肝细胞生长因子联合成纤维细胞生长因子-4诱导人骨髓来源的多能成体祖细胞向肝样细胞分化

目的 探索肝细胞生长因子（HGF）-4成纤维生长因子4-（FGF-4）诱导人骨髓来源多能成体祖细胞（hMAPCs）分化为肝细胞的可行性，为肝组织工程提供新的种子细胞来源。方法 （1）取志愿者适量骨髓后采用梯度密度离心＋贴壁培养获取骨髓间充质干细胞（MSCs），将MSCs通过CD45、GlyA免疫微磁珠负分选得到hMAPCS。（2）将hMAPCs用HGF＋FGF-4进行诱导分化。实验分组：A组：HGF（20ng／m1）＋（FGF-4）10ng／m1诱导hMAPCS；B组（阳性对照组）：L-02人肝细胞株；C组（阴性对照组）：未加任何诱导因素的hMAPCs。（3）免疫细胞化学鉴定不同诱导分化阶段细胞的白蛋白（A1b）、甲胎蛋白（AFP），细胞角蛋白-18（CK-18）等肝细胞特征的表型变化评计数阳性细胞比率。（4）逆转录-聚合酶链反应检测不同诱导分化阶段细胞的A1b、AFP，CK-18的mRNA转录。（5）Western blot检测诱导分化第21，35天后细胞的A1b表达。结果（1）免疫细胞化学结果：A1b、CK18在诱导组中不同时间段基本为阳性着色；AFP在诱导分化第7天为阳性着色，在诱导第14，21天为阴性着色。（2）逆转录聚合酶链反应结果：作为不成熟肝细胞表型的AFP，在诱导分化的第7天有mRNA阳性表达；作为成熟肝细胞表型的A1b及CK-18，在不同时间段mRNA均为阳性表达。（3）Western blot检测诱导分化第21、35天后细胞的A1b表达。结论hMAPCS在一定诱导条件下具有向肝样细胞分化的潜能。     

系统性红斑狼疮患者骨髓造血干/祖细胞体外培养及其发病机制的研究

目的　比较正常人与系统性红班狼疮 (SLE)患者的造血干 /祖细胞生长情况及从造血干 /祖细胞水平探讨SLE的发病机制。方法　用半固体甲基纤维素集落培养法观察 16例活动期及其中 3例经治疗缓解的SLE患者的粒 巨噬细胞集落 (GM CFU)、红系集落 (E CFU)、巨核细胞集落 (MK CFU)。结果　活动期SLE患者的GM CFU、E CFU、MK CFU分别为 (182± 5 0 )、(5 6± 2 7)、(8± 5 ) ,均明显低于正常对照组 ,缓解期SLE患者的GM CFU、E CFU比活动期的明显增加 ,而MK CFU与活动期比较无明显变化。结论　活动期SLE患者的造血干 /祖细胞增生、分化功能异常 ,缓解后的SLE患者其巨核细胞系恢复迟于粒系、红系 ,SLE的发病可能与造血干 /祖细胞的异常有关     

Supportive care and use of hematopoietic growth factors in myelodysplastic syndromes

Supportive care constitutes the basis of the management of patients with myelodysplastic syndromes (MDS). Appropriate treatment of cytopenia, as well as of other related complications, not only improves quality of life but also may positively affect the overall survival of patients. Anemia is the most common cytopenia in MDS, and the requirement for regular transfusions is a major clinical problem for patients with low-risk MDS. An important therapeutic goal in this patient group is to maintain acceptable hemoglobin levels without transfusions. Today, this goal can be achieved by treatment with erythropoietin (Epo) +/- granulocyte colony-stimulating factor (G-CSF), or by more targeted treatment such as antithymocyte globulin or lenalidomide in around 50% of patients. For the remaining patients, and for those who lose their therapeutic response, chronic transfusion therapy, with or without the addition of chelating agents, is the only option and it is important that this treatment is scheduled to meet the needs of the individual patient. Severe thrombocytopenia has recently been reported to respond to thrombopoietic agents, such as AMG 531.     

FLT3 expression initiates in fully multipotent mouse hematopoietic progenitor cells

Lymphoid-primed multipotent progenitors with down-regulated megakaryocyte-erythroid (MkE) potential are restricted to cells with high levels of cell-surface FLT3 expression, whereas HSCs and MkE progenitors lack detectable cell-surface FLT3. These findings are compatible with FLT3 cell-surface expression not being detectable in the fully multipotent stem/progenitor cell compartment in mice. If so, this process could be distinct from human hematopoiesis, in which FLT3 already is expressed in multipotent stem/progenitor cells. The expression pattern of Flt3 (mRNA) and FLT3 (protein) in multipotent progenitors is of considerable relevance for mouse models in which prognostically important Flt3 mutations are expressed under control of the endogenous mouse Flt3 promoter. Herein, we demonstrate that mouse Flt3 expression initiates in fully multipotent progenitors because in addition to lymphoid and granulocyte-monocyte progenitors, FLT3(-) Mk- and E-restricted downstream progenitors are also highly labeled when Flt3-Cre fate mapping is applied.     

骨髓增生异常综合征骨髓克隆造血细胞高表达WT1

目的:调查骨髓增生异常综合征(MDS)骨髓Wilms'肿瘤基因(WT1)是否高表达于恶性克隆细胞。方法:对18例具有克隆标记MDS的骨髓细胞进行了WT1免疫细胞化学(APAAP,碱性磷酸酶-抗碱性磷酸酶)和DNA荧光原位杂交(FISH)的共检测。并与12例正常献血员进行对照。结果:MDS骨髓细胞明显高表达WT1,与对照比较P<0.05。双重染色的结果显示WT1突出地表达于具有异常染色体的恶性克隆造血细胞,而不是正常杂交信号的残存正常造血细胞(P<0.05)。结论:推测MDS骨髓WT1高表达与细胞增殖状态相关。     

Soluble factor cross-talk between human bone marrow-derived hematopoietic and mesenchymal cells enhances in vitro CFU-F and CFU-O growth and reveals heterogeneity in the mesenchymal progenitor cell compartment

The homeostatic adult bone marrow (BM) is a complex tissue wherein physical and biochemical interactions serve to maintain a balance between the hematopoietic and nonhematopoietic compartments. To focus on soluble factor interactions occurring between mesenchymal and hematopoietic cells, a serum-free adhesion-independent culture system was developed that allows manipulation of the growth of both mesenchymal and hematopoietic human BM-derived progenitors and the balance between these compartments. Factorial experiments demonstrated a role for stem cell factor (SCF) and interleukin 3 (IL-3) in the concomitant growth of hematopoietic (CD45+) and nonhematopoietic (CD45-) cells, as well as their derivatives. Kinetic tracking of IL-3alpha receptor (CD123) and SCF receptor (CD117) expression on a sorted CD45- cell population revealed the emergence of CD45-CD123+ cells capable of osteogenesis. Of the total fibroblast colony-forming units (CFU-Fs) and osteoblast colony-forming units (CFU-O), approximately 24% of CFU-Fs and about 22% of CFU-Os were recovered from this population. Cell-sorting experiments demonstrated that the CD45+ cell population secreted soluble factors that positively affect the survival and proliferation of CFU-Fs and CFU-Os generated from the CD45- cells. Together, our results provide insight into the intercellular cytokine network between hematopoietic and mesenchymal cells and provide a strategy to mutually culture both mesenchymal and hematopoietic cells in a defined scalable bioprocess.     

Thrombopoietin, but not erythropoietin promotes viability and inhibits apoptosis of multipotent murine hematopoietic progenitor cells in vitro

The recently cloned c-mpl ligand, thrombopoietin (Tpo), has been extensively characterized with regard to its ability to stimulate the growth, development, and ploidy of megakaryocyte progenitor cells and platelet production in vitro and in vivo. Primitive hematopoietic progenitors have been shown to express c-mpl, the receptor for Tpo. In the present study, we show that Tpo efficiently promotes the viability of a subpopulation of Lin-Sca-1+ bone marrow progenitor cells. The ability of Tpo to maintain viable Lin-Sca-1+ progenitors was comparable to that of granulocyte colony-stimulating factor and interleukin-1, whereas stem cell factor (SCF) promoted the viability of a higher number of Lin-Sca-1+ progenitor cells when incubated for 40 hours. However, after prolonged (> 40 hours) preincubation, the viability- promoting effect of Tpo was similar to that of SCF. An increased number of progenitors surviving in response to Tpo had megakaryocyte potential (37%), although almost all of the progenitors produced other myeloid cell lineages as well, suggesting that Tpo acts to promote the viability of multipotent progenitors. The ability of Tpo to promote viability of Lin-Sca-1+ progenitor cells was observed when cells were plated at a concentration of 1 cell per well in fetal calf serum- supplemented and serum-depleted medium. Finally, the DNA strand breakage elongation assay showed that Tpo inhibits apoptosis of Lin-Sca- 1+ bone marrow cells. Thus, Tpo has a potent ability to promote the viability and suppress apoptosis of primitive multipotent progenitor cells.     

Effects of direct infusion of bone marrow-derived progenitor cells and indirect mobilization of hematopoietic progenitor cells on atherosclerotic plaque and inflammatory process in atherosclerosis

Background

We sought to investigate the effects of lin −/sca + cells, endothelial progenitor cells (EPCs) and granulocyte colony-stimulating factor (G-CSF) administration on atherosclerotic plaque progression.

Methods

Apolipoprotein E-deficient (apoE^−/−) mice were splenectomized and treated with high-cholesterol diet for 6 weeks in order to induce atherosclerotic plaque development. Bone marrow-derived Lin −/sca-1 + cells were isolated and further cultured to early growth endothelial progenitor cells (EPCs). Mice were divided in four groups (n = 10/group) and received two intravenous injections of 5 × 10⁵ cells (lin −/sca-1 + or EPCs), or granulocyte colony-stimulating factor (G-CSF 100 μg/kg/day) for 7 days or normal saline. The same interventions were administered to animals, which had undergone unilateral hind-limb ischemia. Effects on inflammatory parameters, lesion severity, and atherosclerotic plaque area size were assessed.

Results

The administration of both G-CSF and progenitor cells significantly decreased the levels of IL-6, 6 weeks after the initiation of treatment. Atherosclerotic lesion area was reduced by G-CSF (atherosclerotic plaque area percentage 22.94% ± 3.68, p = 0.001), by lin −/sca-1 + (23.27% ± 5.98, p = 0.002) and cultured EPCs (23.16 ± 4.86%, p = 0.002) compared to control (32.75% ± 7.05). In the atherosclerotic mice that underwent limb ischemia, the atherosclerotic plaque area, was not significantly different between the treatment groups cultured EPCs-treated mice and the control group (p = NS, for all).

Conclusions

Direct infusion of progenitor cells and indirect mobilization of hematopoietic progenitor cells decreased plaque progression and levels of inflammatory molecules in a murine model of atherosclerosis. Treatment with G-CSF, lin −/sca-1 +, or EPCs may exert beneficial effects on vascular inflammation and atherosclerotic plaque progression. However, the effects are diminished in an ischemic setting.