Cellular interactions between 3T3 cells and interleukin-3-dependent multipotent haemopoietic cells: a model system for stromal-cell-mediated haemopoiesis

With the aid of a multipotent stem cell line (FDCP-mix cells) co-cultured with either normal or irradiated Swiss 3T3, cellular interactions between stromal cells and haemopoietic stem cells were studied by electron microscopy and time-lapse video microscopy. When cultured in the presence of interleukin 3 (IL-3) but in the absence of stromal cells, the FDCP-mix cells have a characteristic blast morphology. In the absence of IL-3, the cells die unless they are co-cultured with marrow stromal cells or 3T3 cells. In the latter case, they attach, proliferate, and differentiate on both normal and irradiated Swiss 3T3 cell layers without the addition of extrinsic growth factor (IL-3). At the initial attachment sites of these two cell lines, cellular recognition seemed to be mediated by the formation of microvillus cytoplasmic projections and extracellular matrix. These areas may well be the sites of plasma-membrane-bound signalling/adhesional molecules between the interacting cells.     

Rapamycin inhibits osteoblast proliferation and differentiation in MC3T3-E1 cells and primary mouse bone marrow stromal cells

While the roles of the mammalian target of rapamycin (mTOR) signaling in regulation of cell growth, proliferation, and survival have been well documented in various cell types, its actions in osteoblasts are poorly understood. In this study, we determined the effects of rapamycin, a specific inhibitor of mTOR, on osteoblast proliferation and differentiation using MC3T3-E1 preosteoblastic cells (MC-4) and primary mouse bone marrow stromal cells (BMSCs). Rapamycin significantly inhibited proliferation in both MC-4 cells and BMSCs at a concentration as low as 0.1 nM. Western blot analysis shows that rapamycin treatment markedly reduced levels of cyclin A and D1 protein in both cell types. In differentiating osteoblasts, rapamycin dramatically reduced osteoblast-specific osteocalcin (Ocn), bone sialoprotein (Bsp), and osterix (Osx) mRNA expression, ALP activity, and mineralization capacity. However, the drug treatment had no effect on osteoblast differentiation parameters when the cells were completely differentiated. Importantly, rapamycin markedly reduced levels of Runx2 protein in both proliferating and differentiating but not differentiated osteoblasts. Finally, overexpression of S6K in COS-7 cells significantly increased levels of Runx2 protein and Runx2 activity. Taken together, our studies demonstrate that mTOR signaling affects osteoblast functions by targeting osteoblast proliferation and the early stage of osteoblast differentiation.     

Self-renewal and differentiation of interleukin-3-dependent multipotent stem cells are modulated by stromal cells and serum factors

Interleukin-3 (IL-3)-dependent cell lines (FDCP-mix) were cloned and isolated from long-term bone-marrow cultures infected with src-MoMuLV. These cell lines have many of the characteristics of hematopoietic stem cells. Early isolates of the FDCP-mix cells form spleen colonies in irradiated mice and establish long-term hematopoiesis on irradiated marrow stroma in vitro in the absence of IL-3. These two properties of the cells are lost within 15 weeks of establishing the cell lines, but the cell lines retain their ability to differentiate in a multilineage response to hematopoietic growth factors and to hematopoietic stromal cells, as well as to self-renew in the presence of IL-3. The choice between differentiation and self-renewal in FDCP-mix cells can clearly be modified by culture conditions: in particular, cultures containing horse serum preferentially promote self-renewal, whereas cultures containing fetal calf serum preferentially promote differentiation. The FDCP-mix cell lines are not leukemic, nor do they contain the src oncogene. Their ability to respond to hematopoietic growth factors and stroma in a similar manner to normal hematopoietic cells makes them a valuable model for studying the regulation of hemopoietic cell self-renewal and differentiation.     

Bone marrow stromal cells (bone marrow-derived multipotent mesenchymal stromal cells) for bone tissue engineering: basic science to clinical translation

Bone tissue engineering is a promising field of regenerative medicine in which cultured cells, scaffolds, and osteogenic inductive signals are used to regenerate bone. This technology has already been used in several clinical studies and its efficacy has been reported. In this review, we focus on bone marrow stromal cells, which are the most commonly used cell source for bone tissue engineering. The nature of the cells, suitable culture conditions for bone tissue engineering, and their potential therapeutic applications are reviewed with possible caveats. Furthermore, recent advances in bone marrow stromal cell biology are discussed with reference to clinical translation.     

Collection and culture of alveolar bone marrow multipotent mesenchymal stromal cells from older individuals

In this work, we examined the culture condition of alveolar bone marrow multipotent mesenchymal stromal cells (ABMMSCs), aiming to apply regenerative therapy to older periodontitis patients. To better understand the character of cultured cells from alveolar bone marrow, the expression profiles of well‐known genes and their responses to the induction of osteogenic, chondrogenic, or adipogenic differentiation were examined. Using αMEM‐based culture, ABMMSCs could be obtained from older individuals than in previous reports. Interestingly, ABMMSCs expressing Klf4 were able to differentiate into osteoblasts. The prediction of differentiation potential by Klf4 could be a useful guide for further improvement of the culture conditions required to culture ABMMSCs derived from older individuals. J. Cell. Biochem. 107: 1198–1204, 2009. © 2009 Wiley‐Liss, Inc.     

Space flight effects on haemopoietic stem cells of the bone marrow of rats

Abstract. Changes in the number of haemopoietic stem cells (CFU-s) were studied in rats during the recovery day and selected post-recovery days after an 18–19-day flight on biosatellites Cosmos 936 and Cosmos 1129 . There was a decrease in the CFU-s number of the bone marrow of rats on the recovery day. On the 6th day post-recovery the CFU-s number was still depressed, while on the 25th day post-recovery it was elevated above control value. The differentiation ratio of transplanted bone marrow cells was not altered by space flight.     

Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells

Background

Human multipotent mesenchymal stromal cells (MSC) can be isolated from various tissues including bone marrow. Here, MSC participate as bone lining cells in the formation of the hematopoietic stem cell niche. In this compartment, the oxygen tension is low and oxygen partial pressure is estimated to range from 1% to 7%. We analyzed the effect of low oxygen tensions on human MSC cultured with platelet-lysate supplemented media and assessed proliferation, morphology, chromosomal stability, immunophenotype and plasticity.

Results

After transferring MSC from atmospheric oxygen levels of 21% to 1%, HIF-1α expression was induced, indicating efficient oxygen reduction. Simultaneously, MSC exhibited a significantly different morphology with shorter extensions and broader cell bodies. MSC did not proliferate as rapidly as under 21% oxygen and accumulated in G₁ phase. The immunophenotype, however, was unaffected. Hypoxic stress as well as free oxygen radicals may affect chromosomal stability. However, no chromosomal abnormalities in human MSC under either culture condition were detected using high-resolution matrix-based comparative genomic hybridization. Reduced oxygen tension severely impaired adipogenic and osteogenic differentiation of human MSC. Elevation of oxygen from 1% to 3% restored osteogenic differentiation.

Conclusion

Physiologic oxygen tension during in vitro culture of human MSC slows down cell cycle progression and differentiation. Under physiological conditions this may keep a proportion of MSC in a resting state. Further studies are needed to analyze these aspects of MSC in tissue regeneration.     

Skinny people serum factors promote the differentiation of multipotent stem cells into brown adipose tissue

The original study by Alessio et al reported that skinny people (SP) serum can promote the formation of brown adipocytes, but not the differentiation of white adipocytes. This finding may explain why SP do not often become obese, despite consuming more calories than the body needs. More importantly, they demonstrated that circulating factors in SP serum can promote the expression of UCP-1 protein, thereby reducing fat accumulation. In this study, only male serum samples were evaluated to avoid the interference of sex hormones in experiments, but adult males also synthesize estrogen, which is produced by the cells of the testes. At the same time, adult females secrete androgens, and females synthesize androgens that are mainly produced by the adrenal cortex. We believe that the approach of excluding sex hormone interference by sex selection alone may be flawed, so we comment on the article and debate the statistical analysis of the article.     

Cycloheximide or puromycin can substitute for PDGF in inducing cellular DNA synthesis in quiescent 3T3 cells

A brief exposure of quiescent (Go) Swiss 3T3 mouse fibroblasts to inhibitors of protein synthesis can replace platelet-derived growth factor in the stimulation of cellular DNA synthesis. When 3T3 cells, after a 6 hr exposure to either cycloheximide or puromycin, are incubated with platelet-poor plasma, a significant percentage of cells enters DNA synthesis. Either inhibition of protein synthesis, or platelet poor plasma by themselves are totally ineffective. A possible mechanism by which inhibitors of protein synthesis may initiate cell cycle progression is through the activation of the c-myc gene.     

Clonal proliferation of multipotent stem/progenitor cells in the neonatal and adult salivary glands

Salivary gland stem/progenitor cells are thought to be present in intercalated ductal cells, but the fact is unclear. In this study, we sought to clarify if stem/progenitor cells are present in submandibular glands using colony assay, which is one of the stem cell assay methods. Using a low-density culture of submandibular gland cells of neonatal rats, we developed a novel culture system that promotes single cell colony formation. Average doubling time for the colony-forming cells was 24.7 (SD=+/-7.02)h, indicating high proliferative potency. When epidermal growth factor (EGF) and hepatocyte growth factor (HGF) were added to the medium, the number of clonal colonies increased greater than those cultured without growth factors (13.2+/-4.18 vs. 4.5+/-1.73). The RT-PCR and immunostaining demonstrated expressing acinar, ductal, and myoepithelial cell lineage markers. This study demonstrated the presence of the salivary gland stem/progenitor cells that are highly proliferative and multipotent in salivary glands.     

Self-renewal capacity and clonal succession of haemopoietic stem cells in long-term bone marrow culture

The CFU-s proliferative potential varied greatly during long-term cultivation. Most of the CFU-s in the cultures were represented by cells with low renewal capacity. Pre-CFU-s cells capable of producing multipotential colonies in methylcellulose, which contained CFU-s with a high proliferative potential, were identified in the culture. In cultivation of a mixture of cells of different karyotype their ratio changed rapidly from week to week. The findings were consistent with the hypothesis that haemopoietic stem cells are maintained in the culture by the products of a small number of clones which arise and decline in succession, and that pre-CFU-s, but not the CFU-s themselves, are clonogenic progenitors.     

Osteogenic differentiation of multipotent mesenchymal stromal cells isolated from human bone marrow and subcutaneous adipose tissue

Cellular populations with phenotypes similar to multipotent mesenchymal stromal cells were isolated from two different sources, including human bone marrow (BM) and subcutaneous adipose tissue (SAT). Comparative analysis of the efficiency of differentiation in the direction of osteogenesis has revealed morphological changes confirmed by staining with Alizarin red and von Kossa in bone marrow cells at the 14th day and in adipose tissue cells at the 28th day of cultivation in the medium with inductors. Analysis of expression of the osteopontin, osteocalcin, and bone sialoprotein genes in RT-PCR reactions has detected essential differences in the potential of these cells to differentiate into bone tissue cells. Cells isolated from BM of both the control and experimental groups were positive for octeopontin (OP) on the 14th day. Unlike these cells, in cells isolated from SAT in medium without an inductor, no product of OP gene expression was identified. In the cells subjected to differentiation, OP appeared at day 14. In the BM cells, octeocalcin (OC) was found at the 14th day, while the bone sialoprotein (BS) was found at the 21st day of cultivation in induction medium. In cells isolated from SAT, OC, and BS were not detected, even at the 28th day after the beginning of induction.     

Metabolically active antigen presenting cells are required for human T cell proliferation in response to the superantigen streptococcal M protein

Abstract M protein from type 5 group A streptococci has been identified as a member of the family of polyclonal T cell activators termed superantigens because it preferentially stimulates T cells bearing specific Vβ elements of the T cell receptor (TCR). In this study the molecular and cellular requirements for presentation of this protein to T cells were investigated. Only accessory cells (AC) expressing class II major histocompatibility complex (MHC) molecules were capable of supporting T cell activation in response to a 22 kDa fragment of M protein (pep M). Despite the need for class II elements, processing of pep M5 by the antigen-presenting cells (APC) was not required for T cell proliferation induced by pep M5. Fixation of APC by paraformaldehyde (PF) treatment impaired their ability to induce optimal T cell proliferation in response to pep M5 withouth significantly affecting interleukin (IL-2) production. In contrast, PF-fixation of cells from the B cell lymphoma line, Raji, did not affect their ability to present pep M5 to human T cells. Addition of rIL-1 and IL-6 to PF-treated APC restored pep M5-induced blastogenesis. Our data suggest that pep M5 directly associates with HLA class II molecules forming a complex that can induce IL-2 production but not optimal proliferation by T cells. Additional signals provided by the AC are required to trigger optimal T cell proliferation in response to this superantigen.     

The production of factors regulating the proliferation of haemopoietic spleen colony-forming cells by bone marrow macrophages

Media conditioned by normal murine bone marrow cells contain an inhibitor of haemopoietic spleen colony-forming cell proliferation that is concentrated in a nominal 50-100K fraction. Media conditioned by regenerating marrow cells contain a proliferation-stimulatory activity that is concentrated in a nominal 30-50K fraction. Cell separation experiments demonstrated that the activities are produced by adherent, phagocytic, radioresistant, Thy 1.2- Fc+, F4/80+ cells. Cultured macrophages, obtained from long-term marrow cultures or derived from progenitor cells in methyl cellulose cultures are also capable of producing inhibitory and stimulatory activities. The results are consistent with macrophages being an important source of stem cell proliferation regulators in the bone marrow.