Detection of T-precursor cells in human bone marrow and foetal liver.

Human lymphoid cells that have been incubated with conditioned medium from confluent monolayers of human thymic epithelium (HTCM) show an increase in cells forming rosettes with sheep erythrocytes at 4 degrees C (E-rosettes). Those cells demonstrating this in vitro conversion have been interpreted to be T-cell precursors. Separation of human bone marrow cells on discontinuous bovine serum albumin (BSA) gradients, and on a single-step 23% BSA gradient showed enrichment of these T-precursor cells, not only in bone marrow, but also in human foetal liver cells. Bone marrow precursor cells from a patient with Wiskott-Aldrich syndrome (T-cell deficiency) showed a normal in vitro response to HTCM, but no response was seen in cells from a patient with severe combined immunodeficiency disease.     

Immunoperoxidase detection of myeloid antigens in glycolmethacrylate-embedded human bone marrow

Different methods for fixation and exposure of antigenic determinants were tested for detection of a granulocytic differentiation antigen by the monoclonal antibody L12-2, using an indirect immunoperoxidase method on semi-thin sections of undecalcified, glycolmethacrylate-embedded human bone marrow biopsies. Fixation in Bouin's solution for 3 hr gave a more intense and more homogeneous immunological staining than fixation in absolute methanol, 4% formalin, B5, or Michel's medium, and the morphological detail was excellent. Digestion by pronase or trypsin was required. Coating the glass slides with Alcian blue prevented loss of sections from the slides during the staining procedure. Bouin fixation also made possible detection of two other differentiation antigens expressed in the granulocytic series, using the monoclonal antibodies 1G10 and R1B19. Furthermore, the same technique also permitted detection of factor VIII-RAg in the megakaryocytes, as well as recognition of cells of the erythroid series by use of polyclonal rabbit antisera.     

Fetuin in human bone marrow: detection in foetal tissue and patients with mastocytosis

Fetuin, a foetal protein of unknown function, has been shown to be expressed in both the immune and nervous systems, especially during development. Here, we show for the first time, that fetuin is abundantly present in many cells of the foetal human bone marrow, but is restricted to cells of the monocytic lineage in the adult. Fetuin's immunoreactivity increased considerably in adult human bone marrow in some pathological conditions, particularly in mastocytosis and was also increased in bone marrows in some cases of acute leukaemias, especially in acute myeloid leukaemia. This increase in the presence of fetuin in neoplastic bone marrows is not reflected by an increased level of circulating fetuin. This last observation contradicts earlier suggestions that fetuin is specifically reduced in cancer patients. A consistent increase in fetuin immunoreactivity in bone marrow of most cases of mastocytosis, as demonstrated in this paper, could become a useful tool in the diagnosis of this disease.     

Cryopreservation of human cadaveric bone marrow cells

The viability of cryopreserved human cadaveric bone marrow cells was studied using methylcellulose clonal cell culture assays. This is the first study done to reveal the persistence of hemopoietic proliferative and differentiative functions using the methylcellulose clonal cell assay in cryopreserved human cadaveric bone marrow cells which are several hours postmortem. Studies of cryopreserved human cadaveric bone marrows corresponded with those of murine bone marrows. These results strongly suggest that several hours postmortem human cadaveric bone marrow cells can be cryopreserved and may be useful for transplantation.     

Simultaneous analysis of DNA content and surface antigens in human bone marrow

In order to identify when cellular expansion occurs during hematopoietic maturation, a method was developed for the simultaneous analysis of one or two cell-surface antigens and DNA content on bone marrow cells while preserving their light-scatter properties. Proliferation in a population defined by light-scatter and surface-antigenic characteristics was assessed by measuring the percentage of cells in this population having more than 2C amount of DNA ("proliferation index"). Viable, low-density (1.077 g/cm3), bone marrow cells, stained with monoclonal antibodies conjugated with fluorescein or phycoerythrin, were fixed with paraformaldehyde and subsequently treated with the detergent, Tween 20. The UV-excitable DNA stain Hoechst 33342 was used to quantify DNA content in the cells without interference with immunofluorescence. A FACS IV flow cytometer was used, equipped with the first laser at 488 nm emitting for light scattering and immunofluorescence measurements and the second laser emitting at 360 nm for the Hoechst excitation. The Hoechst uptake was the same for all bone marrow populations, yielding a tight coefficient of variation (CV) (average 5.0%) for the G0/G1 DNA peak. This permitted high sensitivity of cell detection in S, G2, and M phases of the cell cycle, while preserving light-scattering properties of the cells and maintaining cell surface immunofluorescence. The lowest "proliferation index" detected using this technique was 0.08% in a sample obtained from a patient with chronic lymphocytic leukemia. Normal helper T lymphocytes in marrow had approximately 0.5% of the cells in S, G2, or M phase. We show that the erythroid lineage, in the adult normal bone marrow, is the most active in proliferation among all hematopoietic lineages.     

Rhythms in human bone marrow and blood cells

In 24h studies of bone marrow (BM), circadian stage-dependent variations were demonstrated in the proliferative activity of BM cells from subsets of 35 healthy diurnally active men. On an average, the percentage of total BM cells in deoxyribonucleic acid (DNA) synthesis phase was 188% greater at midday than at midnight (circadian rhythm: p = 0.018; acrophase or peak time of 13: 16h). Patients with malignant disease (n = 15) and a normal cortisol circadian rhythm showed higher fractions of BM cells in S-phase at midday. Colony-forming units--granulocyte/macrophage (CFU-GM), an indicator of myeloid progenitor cells, showed the same circadian variation as DNA S-phase (average range of change or ROC = 136%; circadian rhythm: p < 0.001; acrophase of 12:09h). Deoxyribonucleic acid S-phase and CFU-GM in BM both showed a circannual rhythm (p = 0.015 and 0.008) with an identical acrophase of August 12. The daily peak in BM glutathione content, a tripeptide involved in cellular defense against cytotoxic damage, preceded BM proliferative peaks by 4-5 h (ROC = 31-90%; circadian rhythm: p = 0.05; acrophase of 08:30h). Myeloid (ROC = 57%; circadian rhythm: p = 0.056; acrophase at 08:40h) and erythroid (ROC = 26%; circadian rhythm: p = 0.01; acrophase of 13:01h) precursor cells were positively correlated (r = 0.41; p < 0.001), indicating a circadian temporal relationship and equal influence on S-phase of total BM cells. Yield of positive selected CD34+ progenitor stem cells also showed significant circadian variation (ROC = 595%; circadian rhythm: p = 0.02; acrophase of 12:40h). Thus, the temporal synchrony in cell cycling renders BM cells more sensitive at specific times to hematopoietic growth factors and cell cycle-specific cytotoxic drugs. Moreover, proper timing of BM harvesting may improve progenitor cell yield. When using marker rhythms in the blood to allow for individualized timing of BM procedures, the times of low values in white blood corpuscles, neutrophils, and lymphocytes and high values in cortisol were predictive of the times of highest BM erythroid, myeloid, and total S-phase numbers occurring in the following 12 h.     

Endosteal cells of the bone marrow stroma in human iliac bone

The data on histological and electron microscopical investigation of the endosteal cells of the human iliac bone are presented. Three types of stromal elements in the endosteum, differing in their ultrastructural organization have been revealed. In the endosteal areas young hemopoietic cells are present, they are closely connected with the stroma. A suggestion is made on an important role of the endosteum in processes of proliferation and differentiation of hemopoietic predecessors.     

The rate of sister chromatid exchange in normal human bone marrow cells

Summary A ring chromosome 22 is described in a 6-year-old mentally retarded boy, who presented a dysmorphic syndrome. The ring chromosome 22 was inherited from the mother, in whom a 46,XX/46,XX,r(22)/45,XY,-15,-22,+t(15;22)(p11;q11) mosaic karyotype was found, indicating a high degree of instability of the chromosome(s) 22 in this woman.     

Immunoelectron microscopic study of the insoluble antigens of bone marrow cells in rats

An immunoperoxidase study of the localization of insoluble antigens was carried out on the rat bone marrow cells. The effect of different fixatives and inhibitors of endogenous peroxidase on the cell ultrastructure and the preservation of immunoreactivity of the cell antigens. The best results were obtained while fixing with 1% paraformaldehyde and 0.05% glutaraldehyde mixture added with 0.5 saponin and using 10% acetic acid as an inhibitor of endogenous peroxidase. Differences were found in the localization of antigens and the intensity of immunoperoxidase staining in cells of different lines of differentiation and degree of maturity.     

The aggregate nature of human mesenchymal stromal cells in native bone marrow

Background aims. The clinical application of mesenchymal stromal cells (MSC) faces several obstacles, such as the lack of a standard method for direct isolation as well as a low frequency and concern about the safety of their in vitro expansion. Although the density-gradient separation technique is used as the first step in most methods of MSC isolation to enrich mononuclear cells, the efficiency of this method has not so far been examined. This study was designed to address this issue. Methods. Human bone marrow (BM) samples were laid over Ficoll-Paque, and after centrifugation the upper and lower fractions were cultured separately. Surface markers, differentiation potential and the number of emerged cells were determined. Results. The isolated cells from both the upper and lower fractions were characteristic of MSC. Although it is commonly believed that MSC are single suspending mononuclear cells and so are enriched in the upper fraction of Ficoll-Paque after density-gradient separation, our data showed that considerable numbers of these cells were accumulated in the lower fraction. Further data indicated that MSC were actually present as cell aggregates in BM and they could be enriched effectively by a simple filtration method. Conclusions. The aggregate nature of MSC in BM is in agreement with the concept that they are one of the main elements of the hematopoietic stem cell niche. In addition, the simple filtration method proposed here to isolate cell aggregates may provide opportunities for instant stem cell therapy without the need for extensive in vitro expansion.     

Proliferation of human hematopoietic bone marrow cells in simulated microgravity

Expansion and/or maintenance of hematopoietic stem cell (HSC) potential following in vitro culture remains a major obstacle in stem cell biology and bone marrow (BM) transplantation. Several studies suggest that culture of mammalian cells in microgravity (micro-g) may reduce proliferation and differentiation of these cells. We investigated the application of these findings to the field of stem cell biology in the hopes of expanding HSC with minimal loss of hematopoietic function. To this end, BM CD34+ cells were cultured for 4-6 d in rotating wall vessels for simulation of micro-g, and assessed for expansion, cell cycle activation, apoptosis, and hematopoietic potential. While CD34+ cells cultured in normal gravity (1-g) proliferated up to threefold by day 4-6, cells cultured in micro-g did not increase in number. As a possible explanation for this, cells cultured in simulated micro-g were found to exit G0/G1 phase of cell cycle at a slower rate than 1-g controls. When assayed for primitive hematopoietic potential in secondary conventional 1-g long-term cultures, cells from initial micro-g cultures produced greater numbers of cells and progenitors, and for a longer period of time, than cultures initiated with 1-g control cells. Similar low levels of apoptosis and adhesion molecule phenotype in micro-g and 1-g-cultured cells suggested similar growth patterns in the two settings. These data begin to elucidate the effects of micro-g on proliferation of human hematopoietic cells and may be potentially beneficial to the fields of stem cell biology and somatic gene therapy.     

Tissue-engineered bone formation with cryopreserved human bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) have become the main cell source for bone tissue engineering. It has been reported that cryopreserved human MSCs can maintain their potential for proliferation and osteogenic differentiation in vitro. There are, however, no reports on osteogenesis with cryopreserved human MSCs in vivo. The aim of this study was to determine whether cryopreservation had an effect on the proliferation capability and osteogenic differentiation of human MSCs on scaffolds in vitro and in vivo. MSCs were isolated from human bone marrow, cultured in vitro until passage 2, and then frozen and stored at −196 °C in liquid nitrogen with 10% Me₂SO as cryoprotectant for 24 h. The cryopreserved MSCs were then thawed rapidly, seeded onto partially demineralized bone matrix (pDBM) scaffolds and cultured in osteogenic media containing 10 mM sodium β-glycerophosphate, 50 μM l-ascorbic acid, and 10 nM dexamethasone. Non-cryopreserved MSCs seeded onto the pDBM scaffolds were used as control groups. Scanning electronic microscopy (SEM) observation, DNA content assays, and measurements of alkaline phosphatase (ALP) activity and osteocalcin (OCN) content were applied, and the results showed that the proliferation potential and osteogenic differentiation of MSCs on pDBM in vitro were not affected by cryopreservation. After 2 weeks of subculture, the MSCs/pDBM composites were subcutaneously implanted into the athymic mice. The constructs were harvested at 4 and 8 weeks postimplantation, and histological examination showed tissue-engineered bone formation in the pDBM pores in both groups. Based on these results, it can be concluded that cryopreservation allows human MSCs to be available for potential therapeutic use to tissue-engineer bone.     

Xeno-free proliferation of human bone marrow mesenchymal stem cells

The proliferation of human bone marrow mesenchymal stem cells (MSCs) employing xeno-free materials not containing fetal calf serum (FCS) and porcine trypsin was investigated for the regenerative medicine of cartilage using MSCs. Four sequential subcultivations of MSCs using a medium containing 10% FCS and recombinant trypsin (TrypLESelect™) resulted in cell growth comparable to that with porcine trypsin. There was no apparent difference in the cell growth and morphology between two kinds of MSC stored in liquid nitrogen using 10% FCS plus DMSO or serum-free TC protector™. MSCs were isolated from human bone marrow cells, stored in liquid nitrogen, and sequentially subcultivated four times employing conventional materials that included FCS, porcine trypsin, and DMSO, or xeno-free materials that included serum-free medium (MesenCult-XF™), TC protector™ and TrypLESelect™. Cells in the culture using the xeno-free materials maintained typical fibroblast-like morphology and grew more rapidly than the cells in the culture using the conventional materials, while the cell surface markers of MSCs (CD90 and CD166) were well maintained in both cultures. Chondrogenic pellet cultures were carried out using these subcultivated cells and a medium containing TGFβ3 and IGF1. The pellet culture using cells grown with the xeno-free materials showed an apparently higher gene expression of aggrecan, a chondrocyte marker, than the pellet culture using cells grown with the conventional materials. Consequently, MSCs that are isolated, stored, and grown using the xeno-free materials including the serum-free medium (MesenCult-XF™), TC protector™, and recombinant trypsin (TrypLESelect™) might be applicable for regenerative medicine of cartilage.     

Receptor tyrosine kinase expression in human bone marrow stromal cells

Bone marrow stromal cells (BMSCs) are a heterogeneous population of cells derived from colony-forming units-fibroblastic (CFU-Fs). These cells reside in the bone marrow cavity and are capable of differentiating into several cell phenotypes including osteoblasts, chondroblasts, hematopoiesis-supporting stromal cells, and adipocytes. However, the factors that regulate the proliferation and differentiation of the BMSC population are for the most part unknown. Since many members of the receptor tyrosine kinase (RTK) family have been shown to participate in growth control of various mesenchymal cell populations, in this study we examined the expression and function of RTKs in the BMSC population. Degenerate oligonucleotides corresponding to two conserved catalytic domains of the RTK family and RT-PCR were used initially to determine which RTKs are expressed in the human BMSC (hBMSC) system. After subcloning the amplification product generated from mRNA of a multicolony-derived hBMSC strain, PDGF receptor (β), EGF receptor, FGF receptor 1, and Axl were identified by DNA sequencing of 26 bacterial colonies. Furthermore, PDGF and EGF were found to enhance BMSC growth in a dose-dependent manner and to induce tyrosine phosphorylation of intracellular molecules, including the PDGF and EGF receptors themselves, demonstrating the functionality of these receptors. On the other hand, bFGF was found to have little effect on proliferation or tyrosine phosphorylation. Since single colony-derived hBMSC strains are known to vary from one colony to another in colony habit (growth rate and colony structure) and the ability to form bone in vivo, the expression levels of these RTKs were determined in 18 hBMSC clonal strains by semiquantitative RT-PCR and were found to vary from one clonal strain to another. While not absolutely predictive of the osteogenic capacity of individual clonal strains, on average, relatively high levels of PDGF-receptor were found in bone-forming strains, while on average, nonbone-forming strains had relatively high levels of EGF-receptor. Taken together, these results indicate that RTKs play a role in the control of hBMSC proliferation, and that the differential pattern of RTK expression may be useful in correlating the biochemical properties of individual clonal strains with their ability to produce bone in vivo. J. Cell. Physiol. 177:426–438, 1998. © 1998 Wiley-Liss, Inc.     

Normal human bone marrow suppressor cells and in liver cirrhosis

Suppressor properties of bone marrow cells were studied in healthy donors and patients with hepatocirrhosis using the technique registrating the activity of bone marrow B-suppressors by the inhibition of xenogenic target cell proliferation. The activity of bone marrow suppressor cells in patients with various types of hepatocirrhosis was reduced as compared to healthy subjects. In addition, the in vitro spontaneous proliferation level of bone marrow cells in hepatocirrhosis was considerably higher than that of healthy donors. This fact can be possibly attributed to the decline in the number of bone marrow B-suppressors or inhibition of their functional activity in hepatocirrhosis. Peripheral blood lymphocytes of these patients, like the lymphocytes of healthy donors, showed practically no suppressive effect in vitro.