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It is well established that haematopoietic stem and progenitor cells (HSPCs) are generated from a transient subset of specialized endothelial cells termed haemogenic, present in the yolk sac, placenta and aorta, through an endothelial-to-haematopoietic transition (EHT). HSPC generation via EHT is thought to be restricted to the early stages of development. By using experimental embryology and genetic approaches in birds and mice, respectively, we document here the discovery of a bone marrow haemogenic endothelium in the late fetus/young adult. These cells are capable of de novo producing a cohort of HSPCs in situ that harbour a very specific molecular signature close to that of aortic endothelial cells undergoing EHT or their immediate progenies, i.e., recently emerged HSPCs. Taken together, our results reveal that HSPCs can be generated de novo past embryonic stages. Understanding the molecular events controlling this production will be critical for devising innovative therapies.

Similar to other adult tissue stem/progenitor cells, bone marrow mesenchymal stem/stromal cells (BM MSCs) exhibit heterogeneity at the phenotypic level and in terms of proliferation and differentiation potential. In this study such a heterogeneity was reflected by the CD200 protein. We thus characterized CD200(pos) cells sorted from whole BM MSC cultures and we investigated the molecular mechanisms regulating CD200 expression. After sorting, measurement of lineage markers showed that the osteoblastic genes RUNX2 and DLX5 were up-regulated in CD200(pos) cells compared to CD200(neg) fraction. At the functional level, CD200(pos) cells were prone to mineralize the extra-cellular matrix in vitro after sole addition of phosphates. In addition, osteogenic cues generated by bone morphogenetic protein 4 (BMP4) or BMP7 strongly induced CD200 expression. These data suggest that CD200 expression is related to commitment/differentiation towards the osteoblastic lineage. Immunohistochemistry of trephine bone marrow biopsies further corroborates the osteoblastic fate of CD200(pos) cells. However, when dexamethasone was used to direct osteogenic differentiation in vitro, CD200 was consistently down-regulated. As dexamethasone has anti-inflammatory properties, we assessed the effects of different immunological stimuli on CD200 expression. The pro-inflammatory cytokines interleukin-1β and tumour necrosis factor-α increased CD200 membrane expression but down-regulated osteoblastic gene expression suggesting an additional regulatory pathway of CD200 expression. Surprisingly, whatever the context, i.e. pro-inflammatory or pro-osteogenic, CD200 expression was down-regulated when nuclear-factor (NF)-κB was inhibited by chemical or adenoviral agents. In conclusion, CD200 expression by cultured BM MSCs can be induced by both osteogenic and pro-inflammatory cytokines through the same pathway: NF-κB., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Human hematopoietic stem/progenitor cells (HSPCs) can be mobilized into the circulation using granulocyte-colony stimulating factor (G-CSF), for graft collection in view of hematopoietic transplantation. This process has been related to bone marrow (BM) release of serine proteases and of the matrix metalloproteinase-9 (MMP-9). Yet, the role of these mediators in HSC egress from their niches remains questionable, because they are produced by nonstromal cells (mainly neutrophils and monocytes/macrophages) that are not a part of the niche. We show here that the G-CSF receptor (G-CSFR) is expressed by human BM mesenchymal stromal/stem cells (MSCs), and that G-CSF prestimulation of MSCs enhances the in vitro trans-stromal migration of CD34+ cells. Zymography analysis indicates that pro-MMP-2 (but not pro-MMP-9) is expressed in MSCs, and that G-CSF treatment increases its expression and induces its activation at the cell membrane. We further demonstrate that G-CSF-stimulated migration depends on G-CSFR expression and is mediated by a mechanism that involves MMPs. These results suggest a molecular model whereby G-CSF infusion may drive, by the direct action on MSCs, HSPC egress from BM niches via synthesis and activation of MMPs. In this model, MMP-2 instead of MMP-9 is implicated, which constitutes a major difference with mouse mobilization models.

Background: Adipogenesis is the developmental process by which mesenchymal stem cells (MSC) differentiate into pre-adipocytes and adipocytes. The aim of the study was to analyze the developmental strategies of human bone marrow MSC developing into adipocytes over a defined time scale. Here we were particularly interested in differentially expressed transcription factors and biochemical pathways. We studied genome-wide gene expression profiling of human MSC based on an adipogenic differentiation experiment with five different time points (day 0, 1, 3, 7 and 17), which was designed and performed in reference to human fat tissue. For data processing and selection of adipogenic candidate genes, we used the online database SiPaGene for Affymetrix microarray expression data., Results: The mesenchymal stem cell character of human MSC cultures was proven by cell morphology, by flow cytometry analysis and by the ability of the cells to develop into the osteo-, chondro- and adipogenic lineage. Moreover we were able to detect 184 adipogenic candidate genes (85 with increased, 99 with decreased expression) that were differentially expressed during adipogenic development of MSC and/or between MSC and fat tissue in a highly significant way (p < 0.00001). Subsequently, groups of up- or down-regulated genes were formed and analyzed with biochemical and cluster tools. Among the 184 genes, we identified already known transcription factors such as PPARG, C/EBPA and RTXA. Several of the genes could be linked to corresponding biochemical pathways like the adipocyte differentiation, adipocytokine signalling, and lipogenesis pathways. We also identified new candidate genes possibly related to adipogenesis, such as SCARA5, coding for a receptor with a putative transmembrane domain and a collagen-like domain, and MRAP, encoding an endoplasmatic reticulum protein., Conclusions: Comparing differential gene expression profiles of human MSC and native fat cells or tissue allowed us to establish a comprehensive differential kinetic gene expression network of adipogenesis. Based on this, we identified known and unknown genes and biochemical pathways that may be relevant for adipogenic differentiation. Our results encourage further and more focused studies on the functional relevance of particular adipogenic candidate genes.

Genostem (acronym for "Adult mesenchymal stem cells engineering for connective tissue disorders. From the bench to the bed side") has been an European consortium of 30 teams working together on human bone marrow Mesenchymal Stem Cell (MSC) biological properties and repair capacity. Part of Genostem activity has been dedicated to the study of basic issues on undifferentiated MSCs properties and on signalling pathways leading to the differentiation into 3 of the connective tissue lineages, osteoblastic, chondrocytic and tenocytic. We have evidenced that native bone marrow MSCs and stromal cells, forming the niche of hematopoietic stem cells, were the same cellular entity located abluminally from marrow sinus endothelial cells. We have also shown that culture-amplified, clonogenic and highly-proliferative MSCs were bona fide stem cells, sharing with other stem cell types the major attributes of self-renewal and of multipotential priming to the lineages to which they can differentiate (osteoblasts, chondrocytes, adipocytes and vascular smooth muscle cells/pericytes). Extensive transcription profiling and in vitro and in vivo assays were applied to identify genes involved in differentiation. Thus we have described novel factors implicated in osteogenesis (FHL2, ITGA5, Fgf18), chondrogenesis (FOXO1A) and tenogenesis (Smad8). Another part of Genostem activity has been devoted to studies of the repair capacity of MSCs in animal models, a prerequisite for future clinical trials. We have developed novel scaffolds (chitosan, pharmacologically active microcarriers) useful for the repair of both bone and cartilage. Finally and most importantly, we have shown that locally implanted MSCs effectively repair bone, cartilage and tendon.

This review describes the historical emergence of the concept of bone marrow mesenchymal stem cells (MSCs), summarizing data on Wolf and Trentin's hematopoietic inductive microenvironment; Dexter's hematopoiesis-supportive stromal cells; Friedenstein's osteogenic cells; and Pittenger's trilineal osteoblastic, chondrocytic, and adipocytic precursors; to finally introduce the specific bone marrow mesenchymal stem cells with differentiation potential to four lineages (mesenchymal and vascular smooth muscle lineages), and stromal and immunomodulatory capacities. Two points are the object of detailed discussion. The first point envisions the stem cell attributes (multipotentiality, self-renewal, tissue regeneration, population heterogeneity, plasticity, and lineage priming) compared with that of the paradigmatic hematopoietic stem cell. In the second point, we discuss the possible existence of bone marrow cells with greater differentiation potential, eventually pluripotential cells. The latter point raises the issues of cell fusion, reprogramming, or selection under nonstandardized conditions of rare populations of neuroectodermal origin, or of cells that had undergone mesenchymal-to-epithelial transition. In the last section, we review data on MSC senescence and possible malignant transformation secondary to extensive culture, gene transfer of telomerase, or mutations such as leading to Ewing's sarcoma. The set of data leads to the conclusion that bone marrow MSCs constitute a specific adult tissue stem cell population. The multiple characteristics of this stem cell type account for the versatility of the mechanisms of injured tissue repair. Although MSC administration may be extremely useful in a number of clinical applications, their transplantation is not without risks that must not be overlooked when developing cell therapy protocols.

Lineage-priming is a molecular model of stem cell (SC) differentiation in which proliferating SCs express a subset of genes associated to the differentiation pathways to which they can commit. This concept has been developed for hematopoietic SCs, but has been poorly studied for other SC populations. Because the differentiation potential of human bone marrow mesenchymal stem cells (BM MSCs) remains controversial, we have explored the theory of lineage-priming applied to these cells. We show that proliferating primary layers and clones of BM MSCs have precise priming to the osteoblastic (O), chondrocytic (C), adipocytic (A), and the vascular smooth muscle (V) lineages, but not to skeletal muscle, cardiac muscle, hematopoietic, hepatocytic, or neural lineages. Priming was shown both at the mRNA (300 transcripts were evaluated) and the protein level. In particular, the master transactivator proteins PPARG, RUNX2, and SOX9 were coexpressed before differentiation induction in all cells from incipient clones. We further show that MSCs cultured in the presence of inducers differentiate into the lineages for which they are primed. Our data point out to a number of signaling pathways that might be activated in proliferating MSCs and would be responsible for the differentiation and proliferation potential of these cells. Our results extend the notion of lineage-priming and provide the molecular framework for inter-A, -O, -C, -V plasticity of BM MSCs. Our data highlight the use of BM MSCs for the cell therapy of skeletal or vascular disorders, but provide a word of caution about their use in other clinical indications.

Bone marrow (BM) mesenchymal stem cells (MSCs) represent an interesting field of research for their in vitro properties and the in vivo therapeutic applications. In the present study, we compared the clonogenic and differentiation capacity of MSCs present in three BM-derived populations-namely, the CD105(+)/CD45(-) cells, the glycophorin A (GlycoA)(-)/CD45(-) cells, and the BM mononuclear cells (BMMCs)-by growing/expanding clones from single colony-forming unit fibroblasts (CFU-F). We also quantified the Oct-4 and Nanog mRNA in the CD105(+)/CD45(-) and GlycoA(-)/CD45(-) cells to define the fraction containing more immature MSCs. We found that basic-fibroblast growth factor (bFGF) favors the long-term survival and growth of the more immature MSCs but has no significant effect on MSC clonogenic potential. CFU-F number and clone recovery were higher in CD105(+)/CD45(-) compared to GlycoA(-)/CD45(-) (p < 0.0001 and p = 0.0364, respectively) cells or BMMCs (p < 0.0001 and p = 0.0007, respectively). The relative mRNA expression of Oct-4 and Nanog was significantly increased in CD105(+)/CD45(-) compared to GlycoA(-)/CD45(-) cells (p < 0.0001 and p < 0.0001, respectively). No significant difference was found in the immunophenotypic characteristics and differentiation potential of clones derived from all three cellular sources. These data suggest that the CD105(+)/CD45(-) BM cell fraction is enriched in immature MSCs and, accordingly, represents an appropriate source for MSC culture initiation.

The adult bone marrow (BM) has been generally considered to be composed of hematopoietic tissue and the associated supporting stroma. There is accumulating evidence that a subset of multipotential cells exists within the latter compartment, commonly referred to as mesenchymal stem cells (MSCs). These cells can be easily expanded ex vivo and induced to differentiate into skeletal connective tissues, thereby emerging as attractive candidates for various therapeutic applications. Despite the extensive in vitro characterization of MSCs, at present, little is known about their in vivo properties or anatomical location. Here we review the efficacy of the different surface markers used to isolate BM MSCs and highlight current data suggesting that BM MSCs in situ are associated with vascular sinus walls and probably belong to the family of vascular smooth muscle cells.

Expression of "stemness" markers is widely used as a predictor of stem cell properties of mesenchymal stem cells (MSC). Here, we show that bone marrow-derived (BM)-MSC show stem cell-like behavior in vivo; that is, they form ossicles with formation of bone, formation of adipocytes, and establishment of the murine hematopoietic microenvironment. Multipotent umbilical vein-derived stromal cells (UVSC), on the other hand, do not form bone, nor do they give rise to adipocytes in vivo. Despite these differences in stem-cell-like behavior, BM-MSC and UVSC express the two transcripts variants of POU5F1 at a similar level. Also, we found that in BM-MSC and UVSC, POU5F1 is detectable. However, more than 89% of the POU5F1 transcripts correspond to the POU5F1P1, -P3, or -P4 pseudogene. Despite low-level expression of POU5F1, we were unable to precipitate POU5F1 protein in either BM-MSC or UVSC. These results demonstrate that MSC stemness does not correlate to expression of POU5F1 transcripts or its pseudogenes.

Objective: Bone marrow (BM) mesenchymal stem cells (MSCs) are being considered as potential therapeutic agents in various inflammatory autoimmune diseases for their tissue-repair and anti-inflammatory tissue-protective properties. This study investigates the reserves and function, the molecular and proteomic profile and the differentiation potential of BM MSCs in patients with active rheumatoid arthritis (RA)., Methods: We evaluated the frequency of MSCs in the BM mononuclear cell fraction using a limiting dilution assay, the proliferative/clonogenic potential and the capacity of cells to differentiate towards the osteogenic/chondrogenic/adipogenic lineages using appropriate culture conditions. We also assessed the molecular and proteomic characteristics in terms of inflammatory cytokine gene and protein expression, the relative telomere length and the survival characteristics of BM MSCs., Results: MSCs from patients with RA (n = 26) and age- and sex-matched healthy individuals (n = 21) were similar in frequency, differentiation potential, survival, immunophenotypic characteristics, and protein profile. Patient MSCs, however, had impaired clonogenic and proliferative potential in association with premature telomere length loss. Transcriptome analysis revealed differential expression of genes related to cell adhesion processes and cell cycle progression beyond the G1 phase. Previous treatment with methotrexate, corticosteroids, anti-cytokine and biological agents or other disease-modifying anti-inflammatory drugs did not correlate with the clonogenic and proliferative impairment of BM MSCs., Conclusion: In spite of some restrictions related to the impaired clonogenic and proliferative potential, our findings support the use of autologous BM MSCs in RA and may have important implications for the ongoing efforts to repair tissue injury commonly seen in the course of the disease.

Bone marrow-derived mesenchymal stem cells (MSC) are able to differentiate into osteoblasts under appropriate induction. Although MSC-derived osteoblasts are part of the hematopoietic niche, the nature of the stromal component in fetal liver remains elusive. Here, we determined the in vitro osteoblastic differentiation potential of murine clonal fetal liver-derived cells (AFT024, BFC012, 2012) in comparison with bone marrow-derived cell lines (BMC9, BMC10). Bone morphogenetic protein-2 (BMP2) increased alkaline phosphatase (ALP) activity, an early osteoblastic marker, in AFT024 and 2012 cells, whereas dexamethasone had little or no effect. BMP2, but not dexamethasone, increased ALP activity in BMC9 cells, and both inducers increased ALP activity in BMC10 cells. BMP2 increased ALP mRNA in AFT024, 2012 and BMC9 cells. By contrast, ALP was not detected in BMC10 and BFC012 cells. BMP2 and dexamethasone increased osteopontin and osteocalcin mRNA expression in 2012 cells. Furthermore, bone marrow-derived cells showed extensive matrix mineralization, whereas fetal liver-derived cell lines showed no or very limited matrix mineralization capacity. These results indicate that the osteoblast differentiation potential differs in bone marrow and fetal liver-derived cell lines, which may be due to a distinct developmental program or different microenvironment in the two hematopoietic sites., (Copyright 2007 Wiley-Liss, Inc.)

We have studied the plasma membrane protein phenotype of human culture-amplified and native bone marrow mesenchymal stem cells (BM MSCs). We have found, using microarrays and flow cytometry, that cultured cells express specifically 113 transcripts and 17 proteins that were not detected in hematopoietic cells. These antigens define a lineage-homogenous cell population of mesenchymal cells, clearly distinct from the hematopoietic lineages, and distinguishable from other cultured skeletal mesenchymal cells (periosteal cells and synovial fibroblasts). Among the specific membrane proteins present on cultured MSCs, 9 allowed the isolation from BM mononuclear cells of a minute population of native MSCs. The enrichment in colony-forming units-fibroblasts was low for CD49b, CD90, and CD105, but high for CD73, CD130, CD146, CD200, and integrin alphaV/beta5. In addition, the expression of CD73, CD146, and CD200 was down-regulated in differentiated cells. The new marker CD200, because of its specificity and immunomodulatory properties, deserves further in-depth studies.

Background: Mesenchymal stromal cells (MSC) have become the focus of cellular therapeutics but little is known regarding bone marrow (BM) MSC derived from children. As MSC constitute part of BM stroma, we examined their properties in children with hematologic diseases., Methods: BM MSC from children with non-malignant hematologic disorders and acute lymphoblastic leukemia (ALL) were isolated and expanded. MSC were immunophenotypically characterized and their functional characteristics were assessed by CFU-F assay and cell doubling time calculation. Their ability for trilineage differentiation was verified by molecular and histochemical methods. Apoptosis was evaluated and clonal analysis was performed., Results: MSC were isolated from BM of all groups. They acquired the mesenchymal-related markers from the first passage, with a simultaneous decrease of hematopoietic markers. A very low percentage of apoptotic cells was detected in all passages. The proliferative and clonogenic capacity did not differ among groups, with the exception of ALL at diagnosis, in which they were defective. Histochemical and molecular analysis of differentiated MSC yielded characteristics for adipocytes, osteoblasts and chondrocytes. Clonal analysis in a number of BM samples revealed a highly heterogeneous population of cells within each clone., Discussion: MSC from BM of children with hematologic disorders, with the exception of ALL at diagnosis, can be isolated in sufficient number and quality to serve as a potential source for clinical applications.

Adult bone marrow (BM)-derived stem cells, including hematopoietic stem cells (HSCs) and MSCs, represent an important source of cells for the repair of a number of damaged tissues. In contrast to HSCs, the soluble factors able to induce MSC migration have not been extensively studied. In the present work, we compared the in vitro migration capacity of human BM-derived MSCs, preincubated or not with the inflammatory cytokines interleukin 1beta (IL1beta) and tumor necrosis factor alpha (TNFalpha), in response to 16 growth factors (GFs) and chemokines. We show that BM MSCs migrate in response to many chemotactic factors. The GFs platelet-derived growth factor-AB (PDGF-AB) and insulin-like growth factor 1 (IGF-1) are the most potent, whereas the chemokines RANTES, macrophage-derived chemokine (MDC), and stromal-derived factor-1 (SDF-1) have limited effect. Remarkably, preincubation with TNFalpha leads to increased MSC migration toward chemokines, whereas migration toward most GFs is unchanged. Consistent with these results, BM MSCs express the tyrosine kinase receptors PDGF-receptor (R) alpha, PDGF-Rbeta, and IGF-R, as well as the RANTES and MDC receptors CCR2, CCR3, and CCR4 and the SDF-1 receptor CXCR4. TNFalpha increases CCR2, CCR3, and CCR4 expression (as opposed to that of CXCR4), together with RANTES membrane binding. These data indicate that the migration capacity of BM MSCs is under the control of a large range of receptor tyrosine kinase GFs and CC and CXC chemokines. Most chemokines are more effective on TNFalpha-primed cells. Our results suggest that the mobilization of MSCs and their subsequent homing to injured tissues may depend on the systemic and local inflammatory state. Disclosure of potential conflicts of interest is found at the end of this article.

We determined a transcriptional profile specific for clonal stromal mesenchymal stem cells from adult and fetal hematopoietic sites. To identify mesenchymal stem cell-like stromal cell lines, we evaluated the adipocytic, osteoblastic, chondrocytic, and vascular smooth muscle differentiation potential and also the hematopoietic supportive (stromal) capacity of six mouse stromal cell lines from adult bone marrow and day 14.5 fetal liver. We found that two lines were quadripotent and also supported hematopoiesis, BMC9 from bone marrow and AFT024 from fetal liver. We then ascertained the set of genes differentially expressed in the intersection set of AFT024 and BMC9 compared with those expressed in the union set of two negative control lines, 2018 and BFC012 (both from fetal liver); 346 genes were upregulated and 299 downregulated. Using Ingenuity software, we found two major gene networks with highly significant scores. One network contained downregulated genes that are known to be implicated in osteoblastic differentiation, proliferation, or transformation. The other network contained upregulated genes that belonged to two categories, cytoskeletal genes and genes implicated in the transcriptional machinery. The data extend the concept of stromal mesenchymal stem cells to clonal cell populations derived not only from bone marrow but also from fetal liver. The gene networks described should discriminate this cell type from other types of stem cells and help define the stem cell state.

Bone marrow (BM) mesenchymal stem cells (MSCs) are non-hematopoietic cells capable of generating colonies of plastic-adherent marrow mesenchymal cells, each derived from a single cell termed a colony-forming unit fibroblasts (CFU-Fs). In addition to their role in establishing the marrow microenvironment, these cells have been shown to differentiate into several types of mesenchymal and non-mesenchymal lineages. Because of their multipotency, MSCs represent an attractive cellular source in the promising field of cellular therapy. In this chapter, we will focus on culture conditions for human BM MSC expansion and CFU-F assays. We also describe the methodologies to analyze the primary cultures obtained, both at the phenotypic and at the functional levels. Phenotypically, MSCs can be defined with a minimal set of markers as CD31-, CD34-, and CD45-negative cells and CD13-, CD29-, CD73-, CD90-, CD105-, and CD166-positive cells. Functionally, we describe the culture conditions (specific media and cellular preparations) for in vitro differentiation of MSCs into the adipogenic, osteogenic, and chondrogenic lineages. The corresponding colorimetric assays (oil red O, Von Kossa and alizarin red S, and safranin O and alcian blue stains, respectively) are also described.

Human marrow-derived STRO-1+ cells were tested for their ability to differentiate into multiple mesenchymal phenotypes. STRO-1+ cells were isolated from long-term cultures of human marrow cells by magnetic immunobeads linked to the antibody STRO-1. Stromal layers generated from STRO-1+ cells, when cocultured without exogenous cytokines for several weeks with CD34+/CD38+ and CD34+/CD38low cells, were able to induce a 10- to 20-fold increase in colony-forming units, and to sustain cobblestone area colony-forming cells with a distinct time course for the different hematopoietic precursors. A myofibroblastic and adipocytic phenotype was observed in hematopoiesis-supportive stromal cells, as shown by electron microscopy and staining of lipids with Nile red. The adipocytic potential of STRO-1+ cells was further demonstrated by flow cytometry of Nile red O-stained cells cultured in adipocyte-induced medium. The same population of STRO-1+ cells cultured in osteoblast-inducing medium was shown to have osteogenic potential, as demonstrated in vitro by the formation of mineral (von Kossa staining), and in vivo by the formation of bone tissue within porous calcium phosphate ceramics implanted into athymic mice. This population of STRO-1+ cells was also shown to have chondrogenic potential in aggregate cultures which displayed a morphologic appearance of cartilage and stained positively with antibodies specific to type II and type X collagens. These results show that the subset of marrow cells that express the STRO-1 antigen is capable of differentiating into multiple mesenchymal lineages including hematopoiesis-supportive stromal cells with a vascular smooth muscle-like phenotype, adipocytes, osteoblasts and chondrocytes., (Copyright 2002 S. Karger AG, Basel)

Human cytomegalovirus (CMV) infection of bone marrow transplant recipients can cause pancytopenia, as well as life-threatening interstitial pneumonia. CMV replicates actively in bone marrow stromal cells, whereas it remains latent in hematopoietic progenitors. Our aim was to study the influence of CMV infection on adherence of CD34(+) cells to the myofibroblastic component of human bone marrow and examine transmission of virus from myofibroblasts to CD34(+) cells. We show that smooth actin, but not fibronectin, organization is markedly modified by CMV infection of bone marrow stromal myofibroblasts. Nonetheless, CMV infection led to increased adherence of the CD34(+) progenitor cell line, KG1a, relative to adherence to uninfected myofibroblasts from the same donors. Adherence of CD34(+) cells to infected bone marrow myofibroblasts resulted in transfer of virions and viral proteins through close cell-to-cell contacts. This phenomenon may play a role in the pathophysiology of CMV bone marrow infection and in eventual virus dissemination.

The hematopoietic system is the adult cellular model in which the biology of the stem cells is the best known and may be a model for numerous other tissues. This model is theoretically based on a hierarchy of cells, which begin on a stem cell that differentiates into mature cells through a large number of cellular stages including hematopoietic progenitors. Hematopoietic stem cells have three ain properties: (1) Self-renewal capacities. However, in transplantation experiments this property is limited and may be regulated by the stem cell niche. (2) Multipotentiality. Hematopoietic stem cells are capable of differentiation towards all myeloid and lymphoid lineages. However, recent experiments suggest that, like other somatic stem cells, hematopoietic stem cells are capable

We have studied the adhesion of human marrow CD34(+) precursors to stromal layer of nontransformed human marrow myofibroblasts (normal stroma) and to different stromal cell lines immortalized by T (PU-34) or t and T (L88/5, L87/4, L2Ori-, KM-102) oncogenes from simian virus 40 and E6, E7 oncogenes from human papilloma virus 16 (HS-27A, HS-23). Flow cytometry and Western blotting studies showed that cells from all lines were stromal myofibroblasts similar to normal stroma. Using an original method of adhesion measurement, we found that adhesion of CD34(+) cells was significantly increased on PU-34 cell layer as compared to normal stroma (43% vs. 27%) whereas adhesion on HS-27A and HS-23 was significantly decreased (11% and 8.5%, respectively), and adhesion on L88/5, L87/4, KM-102 and L2Ori- was negligible to nil (<6%). Adhesion of CD34(+) cells to stromal layers paralleled the expression of alpha smooth muscle (alphaSM) actin within the microfilaments of the cells from the different lines and was inversely correlated to their anchorage-independent growth in semisolid agar. These data show that adhesion to the stromal layer of CD34(+) cells is related to the alphaSM actin microfilamentous network in marrow myofibroblasts and that transformation can negatively affect this microfilamentous network and therefore adhesion of hematopoietic precursors.

This study describes a human bone marrow endothelial cell culture in which endothelial cells are organized into capillary tubes. These endothelial cells were positive for von Willebrand Factor, expressed CD34, CD31, and L-fucose residues, took up acetylated low-density lipoproteins, contained Weibel-Palade bodies, and were ensheathed in a basal lamina (which included laminin beta1, EDa+ and EDb+ fibronectin, and collagen type iv). Pericytes expressing alpha-smooth muscle (alpha-SM) actin were spatially associated with the capillary tubes and there was a highly significant correlation between the number of capillary tubes and pericytes. In this model, basal angiogenesis was found to be vascular endothelial growth factor (VEGF)-dependent, because neutralization of endogenous VEGF induced a dramatic regression in the number of tubes. However, the presence of alpha-SM actin-expressing pericytes in the linings of endothelial tubes partially prevented the VEGF-neutralized tube regression. We also observed that nitric oxide production contributed to basal angiogenesis and that upregulation of nitric oxide increased the number of tubes. Tube numbers also decreased when antibodies neutralizing the integrin alphavbeta5 were applied to the cultures. Moreover, addition of any of the hematopoietic cytokines, erythropoietin, stem cell factor, granulocytic colony stimulating factor, or granulomonocytic colony stimulating factor induced a highly significant increase in tube formation. When erythropoietin and granulocytic colony stimulating factor were added, this increase was larger than the maximum increase observed with VEGF. Thus, we have described an in vitro model for human bone marrow angiogenesis in which pericytes and basal lamina matrix were associated with endothelial cells and formed fully organized capillary tubes. In this model, cytokines known to regulate hematopoiesis also seemed to be mediators of angiogenesis. This culture system may therefore prove to be a valuable tool for the study of hematopoietic cytokines on angiogenesis.

A Moloney-derived retrovirus containing both LacZ and NeoR genes (G1BgSVNa from Genetic Therapy, Inc.), was used to transduce human and murine bone marrow stromal cells. Different kinds of stromal cells that were able to support hematopoiesis were transduced by incubation for 24 h in the presence of virus-containing supernatant. Semiconfluent layers of MRC-5 (human, myofibroblastic, fetal, pulmonary) and MS-5 (murine, myofibroblastic, medullary) cells were successfully transduced after one 24-h incubation, as demonstrated by G418 resistance and Escherichia coli beta-galactosidase staining. In contrast, human stromal cells, purified from primary confluent layers grown for 3-4 weeks, could not be transduced. However, stromal cells generated after 10-12 days in culture from Stro-1+ and 1B10+ stromal precursors were successfully transduced in the presence of basic fibroblast growth factor. Transduced stromal cells maintained a myofibroblastic phenotype, although with a decreased number of alpha-SM actin-positive microfilaments in MS-5 cells. The ability to support the generation of stroma-adherent colony-forming cells from cocultured cord blood CD34+ cells after 4 weeks in culture was similar before and after transduction and G418 selection. In conclusion, human primary stromal precursors can be efficiently transduced, and the stromal cell phenotype and function are not significantly altered after retroviral-mediated transfer of marker genes.

Haemopoiesis is sustained and preferentially committed to granulomonopoiesis by myoid [corrected] stromal cells generated by colony-derived cell lines (CDCL). Using ELISA and RIA, we studied, in the supernatant of cells from CDCL, the time course of interleukins 3 and 6 (IL-3, IL-6), stem cell factor (SCF), granulocyte-macrophage, granulocyte and macrophage colony stimulating factors (GM-CSF, G-CSF and M-CSF), macrophage-inflammatory protein-1alpha (MIP-1alpha) and transforming growth factor beta1 (TGF beta1). IL-6, GM-CSF, M-CSF and MIP-1alpha were released into the supernatant after medium renewal and, except for M-CSF, addition of IL-1beta. G-CSF was detected only after addition of IL-1beta. SCF, contained in medium, first declined and then increased 24 h after medium renewal. Release of TGF beta1 started 24 h after medium renewal and lasted until day 7. IL-3, provided by horse serum, declined throughout the 7d of observation. In conclusion, stromal cells from CDCL synthesized and released into the supernatant. IL-6, GM-CSF, G-CSF, M-CSF and MIP-1alpha after stimulation by seric factor(s) and/or IL-1beta. TGF beta1 was synthesized and released without any obvious extraneous stimuli. There is no definite argument for synthesis of soluble SCF and IL-3. These data support a model where growth factors increase shortly after medium renewal, and negative regulators take over at a later time.

Nontransformed stromal colony-derived cell lines (CDCLs) consist of a pure stromal cell population that differentiates following a vascular smooth-muscle cell repertoire. Here we study the maintenance of hematopoiesis by this cell population. We show that CDCLs allow the generation for several weeks of stroma-adherent colonies (comprising a cobblestone area) from CD34+, CD34+/CD38+, and CD34+/CD38- cells. Stroma-adherent colony-forming cells (CFCs) from CD34+/CD38- cells reach a maximum at week 4 and limiting dilution analysis gives a frequency of 1 per 10 cells seeded; in contrast to this, CFCs from CD34+/CD38+ cells are optimal by week 2 and the frequency is then only 1 per 120 cells seeded. Stroma-adherent colonies comprise hematopoietic cells from all lineages except the T lymphocytic, with a majority of granulomonocytes. CDCLs also allow the amplification of granulomonocytic colony-forming units (CFU-GMs), since cumulative outputs of CFU-GMs by week 6 are 190 and 8 times that observed at culture inception for the CD34+/CD38- and CD34+/CD38+ cell populations, respectively. Our results suggest that stromal cells from CDCLs allow the maintenance of primitive hematopoietic precursors and induce their proliferation and differentiation. This study underscores the potential role of one of the microenvironmental cell populations, that of myoid cells, in the regulation of hematopoietic precursor behavior.

Nontransformed stromal colony-derived cell lines (CDCLs) consist of a pure stromal cell population that differentiates following a vascular smooth muscle cell repertoire, and whose in vivo counterpart is that of myoid cells found in adult and fetal human bone marrow cords. We studied the cytokine expression by reverse-transcriptase polymerase chain reaction (RT-PCR) from pooled fast-growing clones from 10 different bone marrow samples. RT-PCR indicated that 30 cytokines (out of 42 studied) were expressed by CDCLs (20 after medium renewal and hydrocortisone renewal, three after addition of interleukin I beta (IL-1 beta) and seven in only part of the CDCL layers examined). The cytokines expressed comprised mediators known to be involved in the maintenance of early and late hematopoiesis (IL-1 alpha and IL-beta, IL-6, IL-7, IL-8, IL-11 and IL-13; colony-stimulating factors, thrombopoietin, erythropoietin, stem cell factor, fit 3-ligand, hepatocyte cell growth factor, tumor necrosis factor alpha, leukemia inhibitory factor, transforming growth factors beta 1 and beta 3; and macrophage inflammatory protein 1 alpha), angiogenic factors (fibroblast growth factors 1 and 2, vascular endothelial growth factor) and mediators whose usual target (and source) is the connective tissue-forming cells (platelet-derived growth factor A, epidermal growth factor, transforming growth factors alpha and beta 2, oncostatin M and insulin-like growth factor 1), or neuronal cells (nerve growth factor). The cytokines not expressed were lymphokines (IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, and IL-12 and interferon gamma) or mediators synthesized by macrophages (inhibin, activin, platelet-derived growth factor B, and IL-1 receptor antagonist). This study complements the description of the phenotype of the myoid cells, confirming that these cells are the marrow connective tissue-forming cells; moreover, this work suggests that stromal control of hematopoiesis is multifactorial and that myoid cells are involved in the control of marrow angiogenesis and innervation.

We examined long bones from 42 human embryos and fetuses whose gestational ages ranged from 6 to 28 weeks. Bone rudiment sections were stained using a panel of monoclonal antibodies directed at antigens expressed by hematopoietic cells, endothelial cells, smooth muscle cells, fibro-blasts, and stromal cells, to describe the events preceding and accompanying the onset of hematopoiesis in the diaphyseal region. Five distinct stages were identified. Stage I (6.6 to 8.5 gestational weeks [gw]) was that of entirely cartilaginous rudiments: chondrocytes were dilated and capillaries with CD34+ endothelial cells were observed in the perichondral limb mesenchyme. At stage II (8.5-9 gw) chondrolysis was actively proceeding; numerous CD68+ cells were observed, interspersed within the marrow cavity. Stage III (9 to 10.5 gw) was characterized by the development of the vascular bed in the absence of detectable hematopoiesis. At mid-diaphysis, specific structures that we named primary logettes were discernible; they consisted of small chambers of connective tissue, framed by a loose network of CD45-negative cells organized around an arteriole and limited from the surrounding sinus by a clearcut lining of CD34+ endothelial cells flanked on their abluminal side by alpha SM actin+ myoid cells. Stage IV (10.5-15 gw) was characterized by the onset of hematopoiesis. Hematopoietic cells were found exclusively in the primary logettes that had considerably increased in size. Logettes filled with hematopoietic cells were immersed within large and almost empty vascular sinuses. Logettes were attached by a short pedicle to connective tissue adjacent to bone/cartilage remaining formations; this tissue contained very rare hematopoietic cells. Logettes were few, usually less than 10 per long bone, and found solely in the diaphyseal area. Most hematopoietic cells found inside logettes were CD15+ myelocytes; rarely seen were glycophorin A+ immature erythroblasts and CD34+ nonendothelial cells. Hematopoietic cells within the logettes were in contact with alpha SM actin+ myoid cells and flattened endothelial-like (although consistently CD34-negative), aligned cells limiting small capillary lumina. Stage V (16 gw onward) was that of final organization of the long bones with areas of fully calcified bone and areas of dense hematopoiesis where logettes were no longer visible. This study shows three major features of incipient long bone hematopoiesis: 1) absence of CD34+ hematopoietic precursors before the onset of hematopoiesis and extreme rarity of those in the emerging blood-forming marrow, 2) predominance of granulopoiesis, and 3) exclusive development in specific structures organized by vascular cells. This study also suggests that CD68+ cells are instrumental in the chondrolysis process while vascular cells (endothelial and myoid cells) may be the critical microenvironment at the onset of hematopoiesis.

We report a new method for generating nontransformed human stromal cell lines with a replicative potential of 20 to 25 doublings yielding 10(6) to 3 x 10(7) cells after 4 to 6 weeks. Cells from week-3 to -6 adherent layers of human long-term bone marrow cultures (LTBMC) were plated in methylcellulose in the presence of 20 U/mL interleukin-1 beta (IL-1 beta) and 200 U/mL tumor necrosis factor-alpha (TNF-alpha). After 2 to 3 weeks, we obtained 180 +/- 14 colonies per 10(5) cells seeded. These well-delineated colonies with a dense central core consisted of up to several hundred tightly packed, identical, large refractile cells. Colonies were determined to be clones by sequential examination of the cultures and the linear relationship between the number of colonies counted and cells seeded. Colony-derived cell lines (CDCL) were developed by seeding individual colonies in long-term culture medium (LTCM) supplemented with 20 ng/mL basic fibroblast growth factor (bFGF). The selection of colonies yielding lines with high proliferative capacity was due to the presence of IL-1 beta and TNF-alpha in the semisolid medium. The most effective concentrations for clonal selection were 200 U/mL TNF-alpha and 20 U/mL IL-1 beta. The growth of CDCL in liquid culture depended on the presence of bFGF, with the most effective concentration at 20 ng/mL. CDCL were able to maintain the output of colony-forming units granulocyte/macrophage and burst-forming unit-erythrocyte (CFU-GM and BFU-E) for several weeks from cocultured CD34+ marrow cells. The weekly CFU-GM and BFU-E output from weeks 2-5 was at least the same as observed when using passaged adherent layers. CDCL represent a progenitor cell population for stromal cells that may prove a suitable model for the study of the relationship between marrow stromal cells and hematopoiesis.

In a previous report, we described a method to generate cell lines derived from stromal colonies (colony-derived cell lines [CDCL]). In a first step, colonies were obtained from plating cells from adherent layers of human long-term bone marrow cultures (LTBMC) in methyl-cellulose in the presence of interleukin-1 beta (IL-1 beta) (20 U/mL) and tumor necrosis factor-alpha (TNF-alpha) (200 U/mL). In a second step, cell lines were derived from individual colonies cultured in liquid medium with 20 ng/mL basic fibroblast growth factor (bFGF). In this report, we describe the phenotype of cells from more than 100 cell lines. CDCL did not contain cells of hematopoietic origin, which indicates that the culture system did not allow the growth of hematopoietic precursors. CDCL did not contain endothelial-like cells, similarly to primary adherent layers. CDCL comprised stromal cells, as defined by membrane antigens recognized by monoclonal antibodies 6-19, Stro-1 and 1B10. These cells belonged to the family of connective tissue-forming cells since they synthesized interstitial collagens I, III, and V; nonplasmatic EDa+ and EDb- fibronectin; tenascin; and chondroitin-sulfate. Study of the time course of CDCL showed that the lines differed from one another according to their proliferative capacity: 32% of CDCL grew quickly, yielding about 10,000 cells in 10 days; 36% of CDCL grew slowly, yielding 1000 cells in 10 days; and the remaining 32% had intermediate proliferative capacity. For CDCL with a high proliferative capacity, a distinctive differentiation pattern could be described. At culture, inception cells from the lines were vimentin and laminin. Over time, several cytoskeletal and extracellular matrix (ECM) proteins indicative of vascular smooth muscle differentiation were expressed, including: the alpha-SM-actin isoform; the actin-binding proteins, smooth muscle myosin-heavy chain (SMMHC), SM1; h-caldesmon; calponin; gelsolin; and the ECM proteins, collagen IV and elastin. In full-grown lines, cells were similar to immature, intimal, vascular smooth muscle cells as found beneath the endothelium in adult aortas. Because of the coupling between proliferation and differentiation, the differentiation pattern seems to be under genetic control. However, since the coupling was not stringent during the whole lifespan of the lines, it is possible that cytokines are also involved, ensuring autocrine regulation of CDCL development.

Normal human bone marrow stromal cells (BMSC) were isolated from Dexter-type long-term cultures according to their capacity to adhere to plastic and to their lack of hematopoietic antigens. The BMSC displayed a homogeneous appearance and a myofibroblastic phenotype in culture. The stromal cells (SC) were shown to support the proliferation of purified CD34+ hematopoietic progenitors and permitted us to maintain myeloid cells for several weeks in culture. In addition, the BMSC induced the proliferation of purified CD10+ s mu- fetal BM B-cell precursors (BCP). The capacity of the BMSC to induce the proliferation of early myeloid cells was shared by several other human fibroblastic-like cell types. In contrast, the BMSC were far superior to other adherent cells for induction of BCP proliferation. This capacity was largely mediated by endogenously produced interleukin-7 (IL-7), because it could be inhibited by anti-IL-7 antibody. In line with this finding, addition of IL-7 considerably enhanced BCP proliferation in cocultures with skin fibroblasts or synoviocytes. Thus, production of IL-7 appears to be a critical parameter that determines the ability of fibroblastic-like cells to induce BCP proliferation. Taken together, our data show that normal human myofibroblastic BMSC induce the proliferation of both early myeloid and B-lymphoid cells in the absence of accessory hematopoietic cells. The present system should constitute a model to study interactions between native human BM myofibroblastic stroma and various hematopoietic cell subsets.

Fibronectin is a major component of the extracellular matrix of adherent layers of human long-term marrow cultures where it may stabilize the extracellular matrix network and provide adhesion sites for primitive hemopoietic cells. This study was devised to analyze the role of adherent cell populations in fibronectin synthesis, matrix assembly, and degradation. In cultures performed under the conditions described by Gartner and Kaplan, immunoprecipitation after metabolic labeling showed that adherent cells synthesized a fibronectin variant comprising the EDa domain and lacking the EDb one. Vascular smooth muscle-like stromal cells were the cell subset responsible for this synthesis. Once synthesized by stromal cells, EDa+fibronectin was secreted into the supernatant and incorporated into the extracellular matrix. The cumulation in the extracellular matrix was predominant by weeks 5 and 6 of culture, when a decrease in the stromal cell intracytoplasmic content of fibronectin was observed. Stromal cells from a transformed cell line, L2Ori-, were also able to synthesize the EDa+fibronectin variant, although for these cells the assembly into the extracellular matrix was partly impaired. Besides stromal cells, other cell types participated in fibronectin synthesis: early-adhering granulomonocytic cells and macrophages appearing later in culture were able to synthesize an EDa-, EDb- fibronectin variant, clearly distinct from the EDa+ variant produced by stromal cells. Studies on cultures in which macrophage growth was stimulated at the expense of stromal cells by adding granulocyte-macrophage colony-stimulating factor (50 ng/mL) to the culture medium showed a striking decrease in amounts of fibronectin measured in the adherent layer. This decrease was caused by a lack of incorporation of fibronectin in the extracellular matrix, disclosing a major difference between stromal cells and macrophages in terms of matrix assembly. This study confirms the similarity between stromal cells and vascular smooth muscle cells, because in vivo subendothelial intimal aortic smooth muscle cells and cultured smooth muscle cells from the aortic media express the EDa+, EDb- fibronectin variant. Furthermore, our results suggest that the level of fibronectin in adherent layers is regulated by stromal cells and macrophages. The balance between these two cell populations may therefore be crucial for the local control of hemopoiesis by regulating the extracellular fibronectin available for the adhesion of hematopoietic cells. Our data indicate that it may be essential to study the adhesion of stem cells to EDa+, EDb- fibronectin instead of EDa-, EDb- soluble fibronectin, as found in human plasma.

In human long-term marrow cultures connective tissue-forming stromal cells are an essential cellular component of the adherent layer where granulomonocytic progenitors are generated from week 2 onward. We have previously found that most stromal cells in confluent cultures were stained by monoclonal antibodies directed against smooth muscle-specific actin isoforms. The present study was carried out to evaluate the time course of alpha-SM-positive stromal cells and to search for other cytoskeletal proteins specific for smooth muscle cells. It was found that the expression of alpha-SM in stromal cells was time dependent. Most of the adherent spindle-shaped, vimentin-positive stromal cells observed during the first 2 weeks of culture were alpha-SM negative. On the contrary, from week 3 to week 7, most interdigitated stromal cells contained stress fibers whose backbone was made of alpha-SM-positive microfilaments. In addition, in confluent cultures, other proteins specific for smooth muscle were detected: metavinculin, h-caldesmon, smooth muscle myosin heavy chains, and calponin. This study confirms the similarity between stromal cells and smooth muscle cells. Moreover, our results reveal that cells in vivo with the phenotype closest to that of stromal cells are immature fetal smooth muscle cells and subendothelial intimal smooth muscle cells; a cell subset with limited development following birth but extensively recruited in atherosclerotic lesions. Stromal cells very probably derive from mesenchymal cells that differentiate along this distinctive vascular smooth muscle cell pathway. In humans, this differentiation seems crucial for the maintenance of granulomonopoiesis. These in vitro studies were completed by examination of trephine bone marrow biopsies from adults without hematologic abnormalities. These studies revealed the presence of alpha-SM-positive cells at diverse locations: vascular smooth muscle cells in the media of arteries and arterioles, pericytes lining capillaries, myoid cells lining sinuses at the abluminal side of endothelial cells or found within the hematopoietic logettes, and endosteal cells lining bone trabeculae. More or less mature cells of the granulocytic series were in intimate contact with the thin cytoplasmic extensions of myoid cells. Myoid cells may be the in vivo counterpart of stromal cells with the above-described vascular smooth muscle phenotype.

The aim of this review is to analyze the different ways by which stem cells and microenvironmental cells may interact. Stem cells are defined as immature cells that ensure the continuous renewal of blood cells. This small set of marrow cells comprises different kinds of cells, differing by their degree of maturity, their commitment, self-renewal ability and repopulating capacity. Microenvironmental cells are fixed marrow cells involved in stem cell survival, proliferation and differentiation. In vivo studies on the distribution within spleen or marrow, of stem cells injected to lethally irradiated mice, have suggested that cells of the microenvironment play a significant role in stem cell proliferation and differentiation. This role has been demonstrated using an in vitro model, i.e. the long-term marrow cultures as described in 1976 by M. Dexter. Analysis of stem cell maintenance in this culture system has made it possible to define the different means by which stromal cells and macrophages (the microenvironmental cells) may control stem cell behavior. Different molecules play a critical role: cytokines (growth factors and inhibitors), adhesion molecules (cell adhesion molecules and molecules belonging to the extracellular matrix) and eventually small peptides. It appears nowadays possible to materially represent the hemopoietic niche, whose existence was postulated by R. Schofield 10 years ago for theoretical reasons related to the the physiology of stem cells.

We have investigated the distribution of membrane molecules on CD34+ hematopoietic cells isolated from human bone marrow (BM) and cord blood (CB). A distinct CD10+ population was present in BM, but it was not detected in CB. Most CD34+ CD10+ cells in BM were B-cell precursors (BCP), because they expressed CD19. However, CD40 and CD37 were found on the majority of CD34+ cells from either BM or CB, demonstrating that these antigens are not restricted to B-lineage CD34+ cells. CD40 and CD37 were lost during culture of CD34+ cells in the presence of interleukin 3 (IL-3), indicating transient expression early in myeloid development. CD13 antigen was detected on virtually all CD34+ cells from BM and CB. Accordingly, CD13 was present on CD34+ CD10+ cells, demonstrating that this structure is not restricted to myeloid CD34+ cells. In contrast, myeloid CD33 antigen was not detected on CD34+ CD10+ cells. Expression levels of CD13 and of CD33 were heterogeneous in BM, reflecting diversity within the resident CD34+ population. CD25 and CD71 were found on a proportion of CD34+ cells from either BM or CB and maintained during culture in IL-3, consistent with a distribution on activated cells. Finally, a variety of adhesion receptors were present on CD34+ cells. These included the alpha 4 beta 1 (VLA-4), alpha 5 beta 1 (VLA-5), and alpha L beta 2 (LFA-1) integrins, as well as ICAM-1, LFA-3, H-CAM, and LAM-1. Expression of adhesion receptors was remarkably similar in BM and CB, and it followed an all-or-nothing pattern that failed to delineate CD34+ subsets. Taken together, our data show that although CD34+ cells from BM constitute a more heterogeneous population, resident and circulating CD34+ cells largely display the same cell-surface molecules.

The cytoskeleton of stromal cells from the adherent layer of human Dexter-type cultures has been studied. It was found that the stress fibers contained actin specific for smooth muscle, mainly the alpha SM actin isoform. The intermediate filaments consisted of vimentin, and there were no desmin filaments. This pattern was similar to that of cultured vascular smooth muscle cells. The detectability of the alpha SM actin isoform is coincident with the appearance of stromal cells in long-term marrow cultures and may provide a useful marker for stromal cells. The potential in vivo cellular counterpart for stromal cells generated in the Dexter-type culture system is discussed.

Murine bone marrow was fractionated using a Ficoll-Isopaque continuous linear gradient characterized by an osmolarity of 290 mOsm constant throughout the gradient and a physiological pH of 7.4. The cellular peak detected prior to fractionation by means of a 405 nm light beam served as a guide for determining fraction collection. Under these conditions CFC enrichment was observed constantly for densities lower than that of the cellular peak. In 17 experiments the enrichment factor amounted to 3.6 +/- 1.4. Enrichment appeared to be due to both an increase in CFC concentration and an improvement in CFC cloning efficiency. A correlation between the concentration of CFC and that of undifferentiated blasts was observed. The CFC density distribution was dependent on the cell load. For cell loads lower than 25 x 10(6) the modal density of CFC was within the range 1.0615-1.0715. For cell loads higher than 25 x 10(6) there was a shift of the distribution curve towards higher density. Clu-CFC appeared to be denser than CFC. This increase in density may be due to a maturation process from CFC to Clu-FC as a maturation gradient with increasing density was found for the granulocytic series and for erythroblasts.

Normal values for the colony-forming ability of granulocytic progenitor cells (CFC) were established for 26 samples of normal bone marrow and 8 samples of normal blood cultured in methylcellulose. The concentration of CFC in the marrow was measured classically as the number of CFC per 10(5) nucleated marrow cells plated and, in addition, as the number of CFC per 10(5) precursors (myeloblasts, promyelocytes, and myelocytes) and per 10(5) metamyelocytes. A value approximating the total number of marrow CFC was calculated by using the number of CFC per 10(5) metamyelocytes. The normal value for circulating CFC were determined as number per ml of blood, per kg of body weight, per 10(5) circulating lymphocytes, and per 10(5) circulating granulocytes. The value of these different modes of expression are discussed. Although the values reported are not directly applicable from one laboratory to the next, they may constitute a useful reference point. Furthermore, they show that the statistical variation in the results is sufficiently small to permit the comparison of values found for pathologic samples with those determined for normal samples.

Adherent cells from long-term marrow cultures from 23 individuals were transformed with wild-type simian virus 40 (SV40). After transformation, cloned cell lines were developed that even after rigorous subcloning invariably produced both stromal cells and round cells. The stromal cells expressed cytoskeletal filaments similar to those of long-term marrow culture adherent cells and produced interstitial and basal lamina collagen types. The round cells had the electron microscopic appearance of primitive hematopoietic cells and when examined with cytochemical stains and monoclonal antibodies to hematopoietic differentiation antigens had reaction patterns suggestive of cells from several lineages. Most round cells expressed the pan-hematopoietic T-200 determinant, and lesser percentages expressed the early T cell antigens CD-1 and CD-3, HLA-DR determinants, the monocytic antigen recognized by Leu M3, and the myeloid antigens detected by monoclonal antibodies 1G10 and 12.8. In addition, when plated in semisolid medium in the presence of a source of colony-stimulating activity, up to 11% of the cells formed colonies consisting of blastlike cells that also expressed hematopoietic cell surface determinants. The data suggest that adherent cells in long-term marrow cultures contain a cell that after transformation by SV40 obligately produces cells with hematopoietic as well as stromalike features.

This report describes an IgG1 mouse monoclonal antibody derived after immunization of mice with washed stromal cells from human, long-term bone marrow cultures. The antigen recognized by the antibody (BMS-1) is a carbohydrate-containing prosthetic group that is common to and specific for multiple horse serum proteins. These proteins are avidly ingested by stromal cells and concentrated in endocytic vesicles. Cultured smooth muscle cells took up the horse proteins in a similar manner to marrow stromal cells while cultured marrow fibroblasts, endothelial cells, and hepatoma cells did not. These data indicate that marrow stromal cells specifically accumulate horse serum proteins which might partially explain the horse serum requirement for long-term marrow culture maintenance. The data also suggest further similarities between marrow stromal and smooth muscle cells and additional differences between marrow fibroblasts and marrow stromal cells.