Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.     

Serum-free ex vivo expansion of CD34(+) hematopoietic progenitor cells

In an effort to obtain defined culture conditions for ex vivo expansion of hematopoietic stem and progenitor cells which avoid the supplementation of serum, we cultured human CD34(+) hematopoietic progenitor cells in a chemically defined, serum-free medium in the presence of hematopoietic growth factors (HGFs), stem cell factor (SCF), interleukin (IL)-1beta, IL-3, IL-6, and erythropoietin (EPO). A medium, SFM-1, was prepared according to a protocol previously optimized for semisolid progenitor cell assays containing Iscove's Modified Dulbecco's Medium (IMDM) plus cholesterol, bovine serum albumin, transferrin, nucleotides and nucleosides, insulin, and beta-mercaptoethanol. In static cultures seeded with CD34(+)-enriched progenitor cells isolated from human peripheral blood, a mean 76.6-fold expansion of total nucleated cells and a mean 4.6-fold expansion of colony-forming cells (CFC) was recorded after 14 days. Morphological analysis of the expanded cells revealed formation of myeloid, erythroid, and megakaryocytic cells. Flow cytometric analysis indicated that CD34(+) antigen expressing cells were maintained to a limited degree only, and cell populations expressing surface markers for myeloid (CD33, CD14, and CD15) and megakaryocytic (CD41a) lineages predominated. Within SFM-1, bovine serum albumin (BSA), cholesterin, and transferrin represented the most critical components needed for efficient total cell and CFC expansion. Addition of autologous patient plasma (APP) or fetal calf serum (FCS) to SFM-1 resulted in inferior cell amplification and CFC formation compared to controls in SFM-1, indicating that the components used in SFM-1 could replace exogenous serum. Four commercially available serum-free media resulted in either comparable or lower total cell and CFC yields as SFM-1. The transplantation potential of CD34(+) cells after culture in SFM-1 was assayed using limiting dilution analysis on preformed irradiated bone marrow stroma and revealed maintenance of long-term bone marrow culture initiating cell (LTCIC) levels during the culture period. These data indicate that HGF-supported multilineage ex vivo expansion of human CD34(+) hematopoietic progenitor cells is feasible using an IMDM-based culture medium which contains a restricted number of additives, resulting in analogous or improved yields of both primitive and differentiated cells compared to previously established protocols. We suggest that this culture protocol is of advantage when working with pharmaceutical-grade preparations under serum-free conditions.     

Embryonic Hematopoietic Progenitor Cells Reside in Muscle before Bone Marrow Hematopoiesis

In mice, hematopoietic cells home to bone marrow from fetal liver prenatally. To elucidate mechanisms underlying homing, we performed immunohistochemistry with the hematopoietic cell marker c-Kit, and observed c-Kit(+) cells localized inside muscle surrounding bone after 14.5 days post coitum. Flow cytometric analysis showed that CD45(+) c-Kit(+) hematopoietic cells were more abundant in muscle than in bone marrow between 14.5 and 17.5 days post coitum, peaking at 16.5 days post coitum. CD45(+) c-Kit(+) cells in muscle at 16.5 days post coitum exhibited higher expression of Gata2, among several hematopoietic genes, than did fetal liver or bone marrow cells. Colony formation assays revealed that muscle hematopoietic cells possess hematopoietic progenitor activity. Furthermore, exo utero transplantation revealed that fetal liver hematopoietic progenitor cells home to muscle and then to BM. Our findings demonstrate that hematopoietic progenitor cell homing occurs earlier than previously reported and that hematopoietic progenitor cells reside in muscle tissue before bone marrow hematopoiesis occurs during mouse embryogenesis.     

Human CD34+ HLA-DR- bone marrow cells contain progenitor cells capable of self-renewal, multilineage differentiation, and long-term in vitro hematopoiesis.

Human bone marrow cells expressing CD34 but not HLA-DR were isolated by immunofluorescence flow cytometric cell sorting. These cells contained a hematopoietic cell (CFU-B1) capable of producing, in an in vitro semisolid culture system, blast-cell-containing colonies, which possessed the capacity for self-renewal and commitment to multipotential differentiation. In addition, CD34+ HLA-DR- marrow cells contained primitive megakaryocyte progenitor cells, the burst-forming unit-megakaryocyte (BFU-MK). A subset of CD34+ HLA-DR- marrow cells lacking the expression of CD15 and CD71 was obtained by flow cytometric cell sorting and was capable of sustaining in vitro hematopoiesis in suspension culture for up to 8 weeks in the absence of a preestablished adherent marrow cell layer. The combination of IL-3 + IL-1 alpha and IL-3 + IL-6 sustained proliferation of these cells for 8 weeks, induced maximal cellular expansion, and increased the numbers of assayable progenitor cells. These studies demonstrate that human CD34+ HLA-DR- marrow cells and their subsets contain primitive multipotential hematopoietic cells capable of self-renewal and of differentiation into multiple hematopoietic lineages.     

ARP, a peptide derived from the stress-associated acetylcholinesterase variant, has hematopoietic growth promoting activities

BACKGROUND: Psychological stress induces rapid and long-lasting changes in blood cell composition, implying the existence of stress-induced factors that modulate hematopoiesis. Here we report the involvement of the stress-associated "readthrough" acetylcholinesterase (AChE-R) variant, and its 26 amino acid C-terminal domain (ARP) in hematopoietic stress responses. MATERIALS AND METHODS: We studied the effects of stress, cortisol, antisense oligonucleotides to AChE, and synthetic ARP on peripheral blood cell composition and clonogenic progenitor status in mice under normal and stress conditions, and on purified CD34 cells of human origin. We employed in situ hybridization and immunocytochemical staining to monitor gene expression, and 5-bromo-2-deoxyuridine (BrdU), primary liquid cultures, and clonogenic progenitor assays to correlate AChE-R and ARP with proliferation and differentiation of hematopoietic progenitors. RESULTS: We identified two putative glucocorticoid response elements in the human ACHE gene encoding AChE. In human CD34+ hematopoietic progenitor cells, cortisol elevated AChE-R mRNA levels and promoted hematopoietic expansion. In mice, a small peptide crossreacting with anti-ARP antiserum appeared in serum following forced swim stress. Ex vivo, ARP was more effective than cortisol and equally as effective as stem cell factor in promoting expansion and differentiation of early hematopoietic progenitor cells into myeloid and megakaryocyte lineages. CONCLUSIONS: Our findings attribute a role to AChE-R and ARP in hematopoietic homeostasis following stress, and suggest the use of ARP in clinical settings where ex vivo expansion of progenitor cells is required.     

Human granulocyte colony stimulating factor: effects on human long-term bone marrow cultures

Human granulocyte colony stimulating factor (G-CSF) was previously shown to support the survival and proliferation of early myeloid progenitors (pre-CFU) that are capable of generating more mature CFU-GM progenitor cells. To evaluate the scope of action of G-CSF in the hierarchy of hematopoietic stem cells, we studied the effects of recombinant G-CSF (rhG-CSF) on long-term cultures of normal human bone marrow cells (LTBMC). We found that rhG-CSF predominantly influenced initial cell proliferation and expansion of CFU-GM progenitor cells in LTBMC before establishment of a confluent adherent layer. In rhG-CSF-treated LTBMC, the stromal cell layer was associated with a higher proliferative capacity and progenitor cell content as compared to control cultures. This effect was pronounced early after layer confluence and was gradually lost with culture time. rhG-CSF did not alter the duration of the productive phase of LTBMC, suggesting that it may not be active on the hematopoietic stem cells responsible for LTBMC propagation. Alternatively, stromal cells may exert tight regulatory control over progenitor cells, even in the presence of rhG-CSF.     

Umbilical cord blood stem cells can expand hematopoietic and neuroglial progenitors in vitro

The ability of hematopoietic tissue-derived adult stem cells to transdifferentiate into neural progenitor cells offers an interesting alternative to central nervous system (CNS)- or embryonic-derived stem cells as a viable source for cellular therapies applied to brain regeneration. Umbilical cord blood (CB) due to its primitive nature and it unproblematic collection appears as a promising candidate for multipotent stem cell harvest. We developed a negative immunomagnetic selection method that depletes CB from hematopoietic lineage marker-expressing cells, hence isolating a discrete lineage negative (LinNeg) stem cell population (0.1% of CB mononucleated cell [MCN] population). In liquid culture supplemented with thrombopoietin, flt-3 ligand, and c-kit ligand (TPOFLK), CB LinNeg stem cells could expand primitive nonadherent hematopoietic progenitors (up to 47-fold) and simultaneously produce slow-dividing adherent cells with neuroglial progenitor cell morphology over 8 weeks. Laser scanning confocal microscopy analysis identified these adherent cells to express glial fibrillary acidic protein (GFAP). Gene expression analysis showed upregulation of primitive neuroglial progenitor cell markers including, GFAP, nestin, musashi-1, and necdin. ELISA quantification of liquid culture supernatant revealed the in vitro release of transforming growth factor beta-1 (TGFbeta1), glial cell line-derived neurotrophic factor (GDNF) suggesting their contribution to CB LinNeg stem cell transdifferentiation into neuroglial progenitors. Our study supports that a single CB specimen can be pre-expanded in TPOFLK to produce both primitive hematopoietic and neuropoietic progenitors, hence widening CB clinical potential for cellular therapies.     

Collection and analysis of hematopoietic progenitor cells from cynomolgus macaques (Macaca fascicularis): assessment of cross-reacting monoclonal antibodies

Previous studies have shown that hematopoietic progenitor cells can be isolated from human or nonhuman primate bone marrow (BM) cells. In the present study, we studied the cross-reactivity of 13 anti-human CD34, two anti-human c-Kit, and one anti-human CD133 monoclonal antibodies (mAbs) with cynomolgus macaque (Macaca fascicularis) BM cells, using flow cytometric analysis, cell enrichment, and clonogenic assay. Among the 13 anti-human CD34 mAbs assessed, six cross-reacted as previously reported by other groups. However, only three of these six mAbs (clones 561, 563, and 12.8) recognized cynomolgus CD34+ cells that formed progenitor colonies when grown in methylcellulose culture. Similarly, of the two anti-human c-Kit mAbs (clones NU-c-kit and 95C3) that were previously reported to cross-react with cynomolgus BM cells, only one (clone NU-c-kit) resulted in a similar outcome. The anti-human CD133 mAb (clone AC133) also cross-reacted with cynomolgus BM cells, although these cells did not give rise to colonies when grown in culture. These results suggest that antibodies that cross-react with nonhuman primate cells may not identify the hematopoietic cells of interest. In addition, while the CD34 mAb (clone 561) results in the selection of hematopoietic progenitor cells of all lineages when assessed in methylcellulose culture, the c-Kit(high) fraction (NU-c-kit) exclusively identifies erythroid-specific progenitor cells after growth in culture. It is important to consider these findings when selecting cross-reacting mAbs to identify cells of hematopoietic lineages in macaque species.     

Development of a fixed bed bioreactor for the expansion of human hematopoietic progenitor cells

The ex vivo expansion of hematopoietic progenitor cells is of great interest for a variety of clinical applications, e.g. bone marrow transplantation or gene therapy. Therefore it is of general interest to develop a culture system, able to mimic the in vivo hematopoesis, which is a prerequisite for long-term hematopoietic culture. Our approach was to modify a continuously perfused bioreactor for cultivation and expansion of human hematopoietic stem cells. Therefore we immobilized stromal cells (human primary stromal cells or the murine cell line M2-10B4) in porous glass carriers in a fixed bed reactor and cocultivated human hematopoietic progenitor cells for several weeks. After inoculation of mononuclear cells derived from umbilical cord blood or peripheral blood stem cells both adherent and non adherent cells were harvested and analyzed by flow cytometry and short-term colony assays. During cultivation there was a permanent production of progenitor cells and mature blood cells derived from the immobilized cells in the carriers. We could demonstrate the immobilization of hematopoietic progenitor cells of the myeloid system detectable in short-term colony assays. Additionally we could observe the expansion of very early progenitor cells (CFU-GEMM) up to 4.2-fold and later progenitor cells (CFU-GM and BFU-E) up to 7-fold and 1.8-fold, respectively. P.M. and B.S. contributed equal parts to this work. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ex vivo expansion of primitive hematopoietic cells for cellular therapies: An overview

Sources of hematopoietic cells for bone marrow transplantation are limited by the supply of compatible donors, the possibility of viral infection, and autologous (patient) marrow that is depleted from prior chemo- or radiotherapy or has cancerous involvement. Anex vivo system to amplify hematopoietic progenitor cells could increase the number of patients eligible for autologous transplant, allow use of cord blood hematopoietic cells to repopulate an adult, reduce the amount of bone marrow and/or mobilized peripheral blood stem and progenitor cells required for transplantation, and reduce the time to white cell and platelet engraftment. The cloning of hematopoietic growth factors and the identification of appropriate conditions has enabled the development of successfulex vivo hematopoietic cell cultures. Purification systems based on the CD34 marker (which is expressed by the most primitive hematopoietic cells) have proven an essential tool for research and clinical applications. Present methods for hematopoietic cultures (HC) on stromal (i.e. accessory cells that support hematopoiesis) layers in flasks lack a well-controlled growth environment. Several bioreactor configurations have been investigated, and a first generation of reactors and cultures has reached the clinical trial stage. Our research suggests that perfusion conditions improve substantially the performance of hematopoietic reactors. We have designed and tested a perfusion bioreactor system which is suitable for the culture of non-adherent cells (without stromal cells) and readily scaleable for clinical therapies. Eliminating the stromal layer eliminates the need for a stromal cell donor, reduces culture time, and simplifies the culture system. In addition, we have compared the expansion characteristics of both mononuclear and CD34⁺ cells, since the latter are frequently assumed to give a superior performance for likely transplantation therapies.Abbreviations BFU0-E burst forming unit-erythroid - BM bone marrow - CB cord blood - CFU-C colony forming unit-culture - CFU-E colony forming unit-erythroid - CFU-F colony forming unit-fibroblast - CFU-GEMM colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte - CFU-GM colony forming unit-granulocyte, macrophage - CFU-Mix colony forming unit-mixed (also known as CFU-GEMM) - CML chronic myeloid leukemia - CSF colony stimulating factor - DMSO dimethyl sulfoxide - ECM extracellular matrix - EPO erythropoietin - FL fetal liver - HC hematopoietic culture - LTBMC long-term bone marrow culture - LTC-IC long-term culture initiating cell - LTHC long-term hematopoietic culture - MNC mononuclear cells - PB peripheral blood     

Identification of primitive hematopoietic progenitor cells in the rhesus macaque

The close phylogenetic relationship of macaques to humans has resulted in their widespread use as a preclinical model for bone marrow transplantation and stem cell gene therapy. To facilitate further use of this model, we undertook analysis of hematopoietic cells using multiparametric flow cytometric analysis. Rhesus CD34+CD38- cells displayed a number of characteristics of primitive hematopoietic cells, including low forward and orthogonal scatter and the lack of expression of lineage-specific markers or human lymphocyte antigen-DR. Four-color flow cytometric analysis demonstrated that rhesus CD34+CD38- cells were heterogenous with respect to Thy-1 expression and were CD59dim. Quantitative limiting dilution long-term culture-initiating cell (LTC-IC) analysis demonstrated that CD34+CD38- cells were approximately 150-fold enriched for LTC-IC as compared with unfractionated bone marrow, and occurred at a frequency similar to that previously reported in humans. Thus, as in humans, the CD34+38- population of rhesus macaque bone marrow is enriched for primitive, multipotent hematopoietic progenitor cells.     

Limiting factors in murine hematopoietic stem cell assays

Hematopoiesis arguably provides the most well-defined role of stem cells in tissue development, maintenance, and repair, largely because of the experimental methods developed over decades of investigation. Assays of hematopoietic stem and progenitor cell potential were developed in the late 1950s-1960s with the first reports of in vivo transplantation into lethally irradiated recipients (Ford et al., 1956; McCulloch and Till, 1960) and clonal growth of hematopoietic bone marrow cells in vitro (Bradley and Metcalf, 1966). These two major assays have undergone substantial refinement but remain the foundation for defining hematopoietic stem cell biology. Here, we provide a brief overview of methods commonly used to analyze hematopoietic stem and progenitor cell content in mice, discuss the limitations of these assays, and provide an in-depth review of the limiting dilution assay (Szilvassy et al., 1990), the best single assay for quantitating HSC content.     

Comparative distribution of marrow CFU-e and CFU-gm progenitors in different anatomic sites in the dog

The interpretation of marrow cloning activity, particularly in serial cultures, is greatly influenced by the reproducibility of the collected marrow samples. In order to establish whether bone marrow cloning activities and precision of the cloning assays are influenced by the site of bone marrow collection in the dog, we studied the incidence of marrow erythroid (CFU-e) and granulocyte-macrophage (CFU-gm) progenitor cells in the iliac crest, sternum, vertebrae, femur, and humerus, using microplasma clot and soft agar culture systems. Marrow samples obtained from the femur and humerus revealed consistently higher cell concentrations than those from the iliac crest, vertebrae, or sternum. Those aspirated from the sternum and vertebrae had lower cell concentrations and were less reproducible. Statistical analysis revealed no significant differences in the incidence of marrow CFU-e and CFU-gm progenitor cells between the femur, humerus, iliac crest or vertebrae. With multiple sampling, the marrow cloning efficiency was consistent and reproducible within the individual dogs. We conclude that the distribution of CFU-e and CFU-gm is comparable throughout the active marrow in the dog and that these sites may be used interchangeably for multiple quantitative analysis of marrow hematopoietic progenitor cells.     

Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells

Mesenchymal stem cells (MSCs) are a population of pluripotent cells within the bone marrow microenvironment defined by their ability to differentiate into cells of the osteogenic, chondrogenic, tendonogenic, adipogenic, and myogenic lineages. We have developed methodologies to isolate and culture-expand MSCs from human bone marrow, and in this study, we examined the MSC's role as a stromal cell precursor capable of supporting hematopoietic differentiation in vitro. We examined the morphology, phenotype, and in vitro function of cultures of MSCs and traditional marrow-derived stromal cells (MDSCs) from the same marrow sample. MSCs are morphologically distinct from MDSC cultures, and flow cytometric analyses show that MSCs are a homogeneous cell population devoid of hematopoietic cells. RT-PCR analysis of cytokine and growth factor mRNA in MSCs and MDSCs revealed a very similar pattern of mRNAs including IL-6, -7, -8, -11, -12, -14, and -15, M-CSF, Flt-3 ligand, and SCF. Steady-state levels of IL-11 and IL-12 mRNA were found to be greater in MSCs. Addition of IL-1α induced steady-state levels of G-CSF and GM-CSF mRNA in both cell preparations. In contrast, IL-1α induced IL-1α and LIF mRNA levels only in MSCs, further emphasizing phenotypic differences between MSCs and MDSCs. In long-term bone marrow culture (LTBMC), MSCs maintained the hematopoietic differentiation of CD34⁺ hematopoietic progenitor cells. Together, these data suggest that MSCs represent an important cellular component of the bone marrow microenvironment. J. Cell. Physiol. 176:57–66, 1998. © 1998 Wiley-Liss, Inc.     

Human bone marrow CFU-GM and BFU-E localized by light scatter cell sorting

Flow cytometric separation was performed on the normal human bone marrow (BM) by using the low-angle (0 degrees) or high-angle (90 degrees) light scatter. Four distinct subpopulations of cells can be enriched from normal human BM and these fractions were subsequently evaluated for their morphological properties as well as their clonogenic capacity in various progenitor cell assays. Our results indicate that human erythroid and granulocyte-macrophage progenitor cells can be separated from BM low-density cells by cell sorting, and these cells show similar 0 degrees and 90 degrees light scatter properties to those observed with murine bone marrow studies. Flow cytometric analysis also suggests that the majority of sorted BFU-E and CFU-GM resides in the blast cell subset of human BM mononuclear cells.     

High-Density Microwell Chip for Culture and Analysis of Stem Cells

With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)-differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. For example, screening of 700,000 substances would take less than two days, using this platform combined with a conventional bio-imaging system. The microwell chip has standard slide format and consists of 672 wells in total. Each well holds 500 nl, a volume small enough to drastically decrease reagent costs but large enough to allow utilization of standard laboratory equipment. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to either maintain the cells as stem/progenitor cells or to study cell differentiation of stem/progenitor cells over time is demonstrated. Clonality is critical for stem cell research, and was accomplished in the microwell chips by isolation and clonal analysis of single mouse embryonic stem cells using flow cytometric cell-sorting. Protocols for practical handling of the microwell chips are presented, describing a rapid and user-friendly method for the simultaneous study of thousands of stem cell cultures in small microwells. This microwell chip has high potential for a wide range of applications, for example directed differentiation assays and screening of reprogramming factors, opening up considerable opportunities in the stem cell field.     

低氧对CD34^＋造血干／祖细胞增殖分化及其对细胞因子依赖性的影响

To elucidate the effects of hypoxia on the proliferation and differentiation of CD34(+) hematopoietic stem/progenitor cells and their response to cytokines, the cells were isolated from umbilical cord blood by using a high-gradient magnetic cell sorting system (MACS). Mononuclear cells (MNC) and CD34(+) cells were incubated in severe hypoxia (1% oxygen) culture system, and the colony forming cells and antigen expression were studied by colony forming assays and FACS analysis. The results showed that incubation in severe hypoxia increased the number of erythroid bursts (BFU-E) (324.8+/-41.4/10(4) cells) generated from CD34(+) cells (191.2+/-34.5/10(4) cells in the control group, P<0.01). Severe hypoxia also enhanced the maintenance and cloning efficiency of BFU-E in a liquid culture system without growth factors, the number of BFU-E (152.4+/-22.6/10(4)cells) was much bigger than that in the control group (74.2+/-9.3/10(4) cells, P<0.01). In cultures incubated in hypoxia, the percentage of CD34(+) cells was significantly higher (2.5+/-1.2-fold, P<0.05) than in those incubated in air. BFU-E cloning efficiency of MNC was not significantly affected by hypoxia. The above results show that hypoxia enhances the maintenance of erythroid progenitor cells generated from CD34(+) hematopoietic stem/progenitor cells, no matter growth factors are present or not. These positive effects of hypoxia did not occur for the other progenitors.     

A genetic analysis of neural progenitor differentiation

Genetic mechanisms regulating CNS progenitor function and differentiation are not well understood. We have used microarrays derived from a representational difference analysis (RDA) subtraction in a heterogeneous stem cell culture system to systematically study the gene expression patterns of CNS progenitors. This analysis identified both known and novel genes enriched in progenitor cultures. In situ hybridization in a subset of clones demonstrated that many of these genes were expressed preferentially in germinal zones, some showing distinct ventricular or subventricular zone labeling. Several genes were also enriched in hematopoietic stem cells, suggesting an overlap of gene expression in neural and hematopoietic progenitors. This combination of methods demonstrates the power of using custom microarrays derived from RDA-subtracted libraries for both gene discovery and gene expression analysis in the central nervous system.     

Tenascin is a cytoadhesive extracellular matrix component of the human hematopoietic microenvironment

Tenascin is a large extracellular matrix (ECM) glycoprotein found in restricted tissue locations in the adult organism. It is copiously synthesized in regenerative organs or regenerating tissues and by certain tumors. We have analyzed the expression of tenascin in human long term bone marrow cultures as well as in cryostat sections of native bone marrow and found it strongly expressed by the stromal cells of the microenvironment. Two different protein subunits of 280 and 220 kD were detected by immunoblotting. These two forms are derived most likely from two different mRNA splice variants of 6 and 8 kb detected by Northern blotting. The in vivo analysis of cryostat sections showed a codistribution with other ECM molecules such as fibronectin and collagen type III in the microenvironment surrounding the maturing hematopoietic cells. Using two independent cell adhesion assays tenascin could be shown to function as a cytoadhesive molecule for hematopoietic cells. These data suggest a direct involvement of tenascin in the retention of hematopoietic progenitor cells in the stroma.