Alkaline phosphatase, glutathione-S-transferase-P, and cofilin-1 distinguish multipotent mesenchymal stromal cell lines derived from the bone marrow versus peripheral blood.

Multipotent mesenchymal stromal cells (MSCs) can be isolated from bone marrow or peripheral blood. To identify phenotypical and functional differences between MSCs derived from these sources, the human bone marrow-derived, fibroblast-like cell line L87/4 was compared with the peripheral blood-derived, fibroblast-like cell line V54/2. Both cell lines expressed similar levels of SH3+, CD45(-), CD68(-), CD133(-), and HLA-DR(-). The bone marrow-derived cells expressed higher surface levels of CD105, CD10, and CD117 and preferentially expressed alkaline phosphatase, glutathione S-transferase P, and cofilin-1. The peripheral blood-derived line showed a higher number of CD34+/CD105+ double-positive and side population (SP) cells. The results demonstrate the more multipotent, yet quiescent, stromal phenotype of bone marrow MSCs, whereas MSCs isolated from the circulation display more hematopoietic-lineage characteristics. Importantly, potential marker genes that distinguish the two stages of MSCs are defined.

Nonmyeloablative stem cell therapy enhances microcirculation and tissue regeneration in murine inflammatory bowel disease.

BACKGROUND AND AIMS: Reduced microcirculation has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Stem cells or endothelial progenitor cells are thought to contribute to tissue regeneration through neoangiogenesis or vasculogenesis in ischemia- or inflammatory-related diseases. We therefore hypothesized that adult stem cells facilitate epithelial repair in IBD. METHODS: Moderate-severe colitis in mice was induced by dextran sulfate sodium (DSS) and 2.0 x 10(6) immortalized CD34(-) stem cells infused twice via the tail vein during an observation period of 35 days in a nonmyeloablative setting. RESULTS: Here, we demonstrate that adult stem cells home to the damaged digestive tract in the large intestine and facilitate mucosal repair in moderate-severe colitis. Nonmyeloablative stem cell therapy resulted in increased survival in severe colitis (P < .0001). Moreover, clinical activity and histologic evaluation of the colitis severity score were reduced significantly in moderate (P = .0003 or P = .03) and severe (P < .0001 or P < .03) colitis after 35 days, in addition to the DSS-induced shortening of colon length (P = .002 and P < .0002). Genetically marked stem cells were detected predominantly in the submucosa of the damaged colon epithelium. Epithelial repair in experimental IBD was mediated either by induction of improved vasculogenesis or by the differentiation of the transplanted stem cells into endothelial cells, as demonstrated by the promotion of Tie2 activity in the infused cells at the site of the damaged mucosa. CONCLUSIONS: Our findings indicate that systemically administered adult stem cells respond to an adequate tissue lesion in murine IBD by enhancing microcirculation, resulting in accelerated tissue repair.

Milieu-adopted in vitro and in vivo differentiation of mesenchymal tissues derived from different adult human CD34-negative progenitor cell clones.

Adult mesenchymal stem cells with multilineage differentiation potentially exist in the bone marrow, but have also been isolated from the peripheral blood. The differentiation of stem cells after leaving their niches depends predominately on the local milieu and its new microenvironment, and is facilitated by soluble factors but also by the close cell-cell interaction in a three-dimensional tissue or organ system. We have isolated CD34-negative, mesenchymal stem cell lines from human bone marrow and peripheral blood and generated monoclonal cell populations after immortalization with the SV40 large T-antigen. The cultivation of those adult stem cell clones in an especially designed in vitro environment, including self-constructed glass capillaries with defined growth conditions, leads to the spontaneous establishment of pleomorphic three-dimensional cell aggregates (spheroids) from the monoclonal cell population, which consist of cells with an osteoblast phenotype and areas of mineralization along with well-vascularized tissue areas. Modifications of the culture conditions favored areas of bone-like calcifications. After the transplantation of the at least partly mineralized human spheroids into different murine soft tissue sites but also a dorsal skinfold chamber, no further bone formation could be observed, but angiogenesis and neovessel formation prevailed instead, enabling the transplanted cells and cell aggregates to survive. This study provides evidence that even monoclonal adult human CD34-negative stem cells from the bone marrow as well as peripheral blood can potentially differentiate into different mesenchymal tissues depending on the local milieu and responding to the needs within the microenvironment.

Stroma-mediated dysregulation of myelopoiesis in mice lacking I kappa B alpha.

Hematopoiesis occurs in the liver and the bone marrow (BM) during murine development. Newborn mice with a ubiquitous deletion of I kappa B alpha develop a severe hematological disorder characterized by an increase of granulocyte/erythroid/monocyte/macrophage colony-forming units (CFU-GEMM) and hypergranulopoiesis. Here, we report that this particular myeloproliferative disturbance is mediated by continuously deregulated perinatal expression of Jagged1 in I kappa B alpha-deficient hepatocytes. The result is a permanent activation of Notch1 in neutrophils. In contrast, in mice with a conditional deletion of I kappa B alpha only in the myeloid lineage (ikba(flox/flox) x LysM-Cre) and in fetal liver cell chimeras (ikba(FL delta/FL delta)), a cell-autonomous induction of the myeloproliferative disease was not observed. Coculture of I kappa B alpha-deficient hepatocytes with wild-type (wt) BM cells induced a Jagged1-dependent increase in CFUs. In summary, we show that cell-fate decisions leading to a premalignant hematopoietic disorder can be initiated by nonhematopoietic cells with inactive I kappa B alpha.

Improved arteriogenesis with simultaneous skeletal muscle repair in ischemic tissue by SCL(+) multipotent adult progenitor cell clones from peripheral blood.

BACKGROUND: The CD34(-) murine stem cell line RM26 cloned from peripheral blood mononuclear cells has been shown to generate hematopoietic progeny in lethally irradiated animals. The peripheral blood-derived cell clones expresses a variety of mesodermal and erythroid/myeloid transcription factors suggesting a multipotent differentiation potential like the bone marrow-derived 'multipotent adult progenitor cells' (MAP-C). METHODS: SCL(+) CD34(-) RM26 cells were transfused intravenously into mice suffering from chronic hind-limb ischemia, evaluating the effect of stem cells on collateral artery growth and simultaneous skeletal muscle repair. RESULTS: RM26 cells are capable of differentiating in vitro into endothelial cells when cultured on the appropriate collagen matrix. Activation of the SCL stem cell enhancer (SCL(+)) is mediated through the binding to two Ets and one GATA site and cells start to express milieu- and growth condition-dependent levels of the endothelial markers CD31 (PECAM) and Flt-1 (VEGF-R1). Intravenously infused RM26 cells significantly improved the collateral blood flow (arteriogenesis) and neo-angiogenesis formation in a murine hind-limb ischemia transplant model. Although transplanted RM26 cells did not integrate into the growing collateral arteries, cells were found adjacent to local arteriogenesis, but instead integrated into the ischemic skeletal muscle exclusively in the affected limb for simultaneous tissue repair. CONCLUSION: These data suggest that molecularly primed hem-/mesangioblast-type adult progenitor cells can circulate in the peripheral blood improving perfusion of tissues with chronic ischemia and extending beyond the vascular compartment.

The co-expression of CD117 (c-kit) and osteocalcin in activated bone marrow stem cells in different diseases.

It is still difficult to identify a potential stem cell in the bone marrow which can give rise to haematopoiesis and mesenchymal cells. In the past, the stem cells for both tissues were considered to be from different stem cell pools, but it has been shown recently in vitro and in vivo that there is an unexpected plasticity at least among early haemopoietic progenitors which can give rise also to mesenchymal tissue. In an attempt to identify stem cells in the bone marrow, which are common precursors to both lineages, we observed that fibroblast-like periosteal cells changed their morphology towards an osteoblastic differentiation with a more cuboidal or triangular morphology especially close to metastatic infiltrates. As a marker for haemopoietic progenitors, we used an antibody against the tyrosine kinase receptor c-kit (CD117) and an anti-osteocalcin antibody to stain mesenchymal cells with a osteoblastic potential. Normal bone marrow specimens only showed a discrete expression pattern of CD117 and osteocalcin, but periosteal stem cells, which strongly co-express the applied haemopoietic and mesenchymal markers, were found particularly in the bone marrow of patients with infiltrates of malignant lymphoma or metastasis from prostate or breast cancer. The evaluation of bone marrow specimens from patients with an aplastic syndrome or myelodysplastic syndrome showed a more heterogenous expression pattern. Our results show that a stem cell candidate common to haematopoiesis and mesenchymal progeny can be detected in bone marrow specimens after activation, as demonstrated by the co-expression of CD117 and osteocalcin, which also seems to be associated with haematological diseases or metastatic infiltration in the bone marrow.