Human blood-mobilized hematopoietic precursors differentiate into osteoclasts in the absence of stromal cells.

Osteoclastogenesis is a complex process that is facilitated by bone marrow stromal cells (SCs). To determine if SCs are an absolute requirement for the differentiation of human hematopoietic precursors into fully mature, osteoclasts (OCs), CD34+ cells were mobilized into the peripheral circulation with granulocyte colony-stimulating factor, harvested by leukapheresis, and purified by magnetic-activated cell sorting. This procedure yields a population of CD34+ cells that does not contain SC precursors, as assessed by the lack of expression of the SC antigen Stro-1, and that differentiates only into hematopoietic cells. We found that CD34+, Stro-1- cells cultured with a combination of granulocyte/macrophage colony-stimulating factor, interleukin 1, and interleukin 3 generated cells that fulfill current criteria for the characterization of OCs, including multinucleation, presence of tartrate-resistant acid phosphatase, and expression of the calcitonin and vitronectin receptors and of pp60c-src tyrosine kinase. These OCs also expressed mRNA for the noninserted isoform of the calcitonin receptor and excavated characteristic resorption pits in devitalized bone slices. These data demonstrate that accessory SCs are not essential for human osteoclastogenesis and that granulocyte colony-stimulating factor treatment mobilizes OC precursors into the peripheral circulation.

Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells

Marrow stromal cells are adult stem cells from bone marrow that can differentiate into multiple nonhematopoietic cell lineages. Previous reports demonstrated that single-cell-derived colonies of marrow stromal cells contained two morphologically distinct cell types: spindle-shaped cells and large flat cells. Here we found that early colonies also contain a third kind of cell: very small round cells that rapidly self-renew. Samples enriched for the small cells had a greater potential for multipotential differentiation than samples enriched for the large cells. Also, the small cells expressed a series of surface epitopes and other proteins that potentially can be used to distinguish the small cells from the large cells. The results suggested it will be important to distinguish the major subpopulations of marrow stromal cells in defining their biology and their potential for cell and gene therapy.

Anti-HIV-1 and chemotactic activities of human stromal cell-derived factor 1α (SDF-1α) and SDF-1β are abolished by CD26/dipeptidyl peptidase IV-mediated cleavage

CD26 is a leukocyte-activation antigen that is expressed on T lymphocytes and macrophages and possesses dipeptidyl peptidase IV (DPPIV) activity, whose natural substrates have not been identified yet. CXC chemokines, stromal cell-derived factor 1α (SDF-1α) and 1β (SDF-1β), sharing the receptor CXCR-4, are highly efficacious chemoattractants for resting lymphocytes and CD34+ progenitor cells, and they efficiently block the CXCR-4-mediated entry into cells of T cell line tropic strains of HIV type 1 (HIV-1). Here we show that both the chemotactic and antiviral activities of these chemokines are abrogated by DPPIV-mediated specific removal of the N-terminal dipeptide, not only when the chemokines are produced in transformed mouse L cell line to express human CD26 but also when they were exposed to a human T cell line (H9) physiologically expressing CD26. Mutagenesis of SDF-1α confirmed the critical requirement of the N-terminal dipeptide for its chemotactic and antiviral activities. These data suggest that CD26-mediated cleavage of SDF-1α and SDF-1β likely occurs in human bodies and promotes HIV-1 replication and disease progression. They may also explain why memory function of CD4+ cells is preferentially lost in HIV-1 infection. Furthermore, CD26 would modulate various other biological processes in which SDF-1α and SDF-1β are involved.

Interleukin 7 independent development of human B cells.

Mammalian hematopoietic stem cell (HSC) commitment and differentiation into lymphoid lineage cells proceed through a series of developmentally restricted progenitor compartments. A complete understanding of this process, and how it differs from HSC commitment and differentiation into cells of the myeloid/erythroid lineages, requires the development of model systems that support HSC commitment to the lymphoid lineages. We now describe a human bone marrow stromal cell culture that preferentially supports commitment and differentiation of human HSC to CD19+ B-lineage cells. Fluorescence activated cell sorterpurified CD34++/lineage-cells were isolated from fetal bone marrow and cultured on human fetal bone marrow stromal cells in serum-free conditions containing no exogenous cytokines. Over a period of 3 weeks, CD34++/lineage- cells underwent commitment, differentiation, and expansion into the B lineage. Progressive changes included: loss of CD34, acquisition of and graded increases in the level of cell surface CD19, and appearance of immature B cells expressing mu/kappa or mu/lambda cell surface Ig receptors. The tempo and phenotype of B-cell development was not influenced by the addition of IL-7 (10 ng/ml), or by the addition of goat anti-IL-7 neutralizing antibody. These results indicate a profound difference between mouse and human in the requirement for IL-7 in normal B-cell development, and provide an experimental system to identify and characterize human bone marrow stromal cell-derived molecules crucial for human B lymphopoiesis.

Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium.

A major goal of experimental and clinical hematology is the identification of mechanisms and conditions that support the expansion of transplantable hematopoietic stem cells. In normal marrow, such cells appear to be identical to (or represent a subset of) a population referred to as long-term-culture-initiating cells (LTC-ICs) so-named because of their ability to produce colony-forming cell (CFC) progeny for > or = 5 weeks when cocultured with stromal fibroblasts. Some expansion of LTC-ICs in vitro has recently been described, but identification of the factors required and whether LTC-IC self-renewal divisions are involved have remained unresolved issues. To address these issues, we examined the maintenance and/or generation of LTC-ICs from single CD34+ CD38- cells cultured for variable periods under different culture conditions. Analysis of the progeny obtained from cultures containing a feeder layer of murine fibroblasts engineered to produce steel factor, interleukin (IL)-3, and granulocyte colony-stimulating factor showed that approximately 20% of the input LTC-ICs (representing approximately 2% of the original CD34+ CD38- cells) executed self-renewal divisions within a 6-week period. Incubation of the same CD34+ CD38- starting populations as single cells in a defined (serum free) liquid medium supplemented with Flt-3 ligand, steel factor, IL-3, IL-6, granulocyte colony-stimulating factor, and nerve growth factor resulted in the proliferation of initial cells to produce clones of from 4 to 1000 cells within 10 days, approximately 40% of which included > or = 1 LTC-IC. In contrast, in similar cultures containing methylcellulose, input LTC-ICs appeared to persist but not divide. Overall the LTC-IC expansion in the liquid cultures was 30-fold in the first 10 days and 50-fold by the end of another 1-3 weeks. Documentation of human LTC-IC self-renewal in vitro and identification of defined conditions that permit their extensive and rapid amplification should facilitate analysis of the molecular mechanisms underlying these processes and their exploitation for a variety of therapeutic applications.

Long-term in vivo expression of the human glucocerebrosidase gene in nonhuman primates after CD34+ hematopoietic cell transduction with cell-free retroviral vector preparations.

Successful gene transfer into stem cells would provide a potentially useful therapeutic modality for treatment of inherited and acquired disorders affecting hematopoietic tissues. Coculture of primate bone marrow cells with retroviral producer cells, autologous stroma, or an engineered stromal cell line expressing human stem cell factor has resulted in a low efficiency of gene transfer as reflected by the presence of 0.1-5% of genetically modified cells in the blood of reconstituted animals. Our experiments in a nonhuman primate model were designed to explore various transduction protocols that did not involve coculture in an effort to define clinically useful conditions and to enhance transduction efficiency of repopulating cells. We report the presence of genetically modified cells at levels ranging from 0.1% (granulocytes) to 14% (B lymphocytes) more than 1 year following reconstitution of myeloablated animals with CD34+ immunoselected cells transduced in suspension culture with cytokines for 4 days with a retrovirus containing the glucocerebrosidase gene. A period of prestimulation for 7 days in the presence of autologous stroma separated from the CD34+ cells by a porous membrane did not appear to enhance transduction efficiency. Infusion of transduced CD34+ cells into animals without myeloablation resulted in only transient appearance of genetically modified cells in peripheral blood. Our results document that retroviral transduction of primate repopulating cells can be achieved without coculture with stroma or producer cells and that the proportion of genetically modified cells may be highest in the B-lymphoid lineage under the given transduction conditions.

Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic–scid/scid mice

Human hematopoiesis originates in a population of stem cells with transplantable lympho-myeloid reconstituting potential, but a method for quantitating such cells has not been available. We now describe a simple assay that meets this need. It is based on the ability of sublethally irradiated immunodeficient nonobese diabetic–scid/scid (NOD/SCID) mice to be engrafted by intravenously injected human hematopoietic cells and uses limiting dilution analysis to measure the frequency of human cells that produce both CD34−CD19+ (B-lymphoid) and CD34+ (myeloid) colony-forming cell progeny in the marrow of such recipients 6 to 8 weeks post-transplant. Human cord blood (CB) contains ≈5 of these competitive repopulating units (CRU) per ml that have a similar distribution between the CD38− and CD38+ subsets of CD34+ CB cells as long-term culture-initiating cells (LTC-IC) (4:1 vs. 2:1). Incubation of purified CD34+CD38− human CB cells in serum-free medium containing flt-3 ligand, Steel factor, interleukin 3, interleukin 6, and granulocyte colony-stimulating factor for 5–8 days resulted in a 100-fold expansion of colony-forming cells, a 4-fold expansion of LTC-IC, and a 2-fold (but significant, P < 0.02) increase in CRU. The culture-derived CRU, like the original CB CRU, generated pluripotent, erythroid, granulopoietic, megakaryopoietic, and pre-B cell progeny upon transplantation into NOD/SCID mice. These findings demonstrate an equivalent phenotypic heterogeneity amongst human CB cells detectable as CRU and LTC-IC. In addition, their similarly modest response to stimulation by a combination of cytokines that extensively amplify LTC-IC from normal adult marrow underscores the importance of ontogeny-dependent changes in human hematopoietic stem cell proliferation and self-renewal.

Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice

The purification of primitive human hematopoietic stem cells has been impaired by the absence of repopulation assays. By using a stringent two-step strategy involving depletion of lineage-positive cells followed by fluorescence-activated cell sorting, we have purified a cell population that is highly enriched for cells capable of multilineage repopulation in nonobese diabetic/severe combined immunodeficient (NOD/SCID) recipients. These SCID-repopulating cells (SRCs) were exclusively found in a cell fraction that expressed high levels of CD34 and no CD38. Through limiting dilution analysis using Poisson statistics, we calculated a frequency of 1 SRC in 617 CD34+ CD38− cells. The highly purified SRC were capable of extensive proliferation in NOD/SCID mice. Mice transplanted with 1 SRC (at limiting cell doses) were able to produce approximately 400,000 progeny 6 weeks after the transplant. Detailed flow cytometric analysis of the marrow of highly engrafted mice demonstrated both lymphoid and myeloid differentiation, as well as the retention of a significant fraction of CD34+ CD38− cells. These highly purified fractions should be useful for identification of the cellular and molecular mechanisms that regulate primitive human hematopoietic cells. Moreover, the ability to detect and purify primitive cells provides a means to develop conditions for maintaining and/or expanding these cells during in vitro culture.

Purified hematopoietic stem cells without facilitating cells can repopulate fully allogeneic recipients across entire major histocompatibility complex transplantation barrier in mice

We report herein the successful long term engraftment of highly purified hematopoietic stem cells (HSCs) without any facilitating cells in fully allogeneic recipient mice across the entire major histocompatibility complex (MHC) transplantation barrier. This finding challenges the assumption that highly purified marrow HSCs alone cannot produce long-lived allogeneic bone marrow chimeras across the MHC barrier. In the present experiments, 1 × 105 HSCs from 5-fluorouracil (5-FU)-treated donors, without any facilitating cells, have been found to repopulate lethally irradiated fully allogeneic recipients. Low density, lineage-negative (CD4−, CD8−, B220−, Mac-1−, Gr-1−), CD71-negative, class I highly positive, FACS-sorted cells from 5-FU-treated C57BL/6 (B6) donor mice were transplanted into lethally irradiated BALB/c recipients. (BALB/c → BALB/c) → BALB/c T cell-depleted marrow cells used as compromised cells were also transplanted into the recipients to permit experiments to be pursued over a long period of time. Cells of donor origin in all recognized lineages of hematopoietic cells developed in these allogeneic chimeras. One thousand HSCs were sufficient to repopulate hemiallogeneic recipients, but 1 × 104 HSCs alone from 5-FU-treated donors failed to repopulate the fully allogeneic recipients. Transplantation of primary marrow stromal cells or bones of the donor strain into recipient, together with 1 × 104 HSCs, also failed to reconstitute fully allogeneic recipients. Suppression of resistance of recipients by thymectomy or injections of granulocyte colony-stimulating factor before stem cell transplantation enhanced the engraftment of allogeneic HSCs. Our experiments show that reconstitution of all lymphohematopoietic lineages across the entire MHC transplantation barriers may be achieved by transplanting allogeneic HSCs alone, without any facilitating cells, as long as a sufficient number of HSCs is transplanted.

Tuning of activation thresholds explains flexibility in the selection and development of T cells in the thymus

Immature CD4+CD8+ thymocytes expressing T-cell antigen receptors (TCR) are selected by TCR-mediated recognition of peptides associated with major histocompatibility complex molecules on thymic stromal cells. Selection ensures reactivity of the mature cells to foreign antigens and tolerance to self. Although much has been learned about the factors that determine whether a thymocyte with a given specificity will be positively or negatively selected, selection as an aspect of the developmental process as a whole is less well-understood. Here we invoke a model in which thymocytes tune their response characteristics individually and dynamically in the course of development. Cellular development and selection are driven by receptor-mediated metabolic perturbations. Perturbation is a measure of the net intracellular change induced by external stimulation. It results from the integration of several signals and countersignals over time and therefore depends on the environment and the maturation stage of the cell. Individual cell adaptation limits the range of perturbations. Such adaptation renders thymocytes less sensitive to the level of stimulation per se, but responsive to environmental changes in that level. This formulation begins to explain the mechanisms that link developmental and selection events to each other.

Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum.

Rare nucleated fetal cells circulate within maternal blood. Noninvasive prenatal diagnosis by isolation and genetic analysis of these cells is currently being undertaken. We sought to determine if genetic evidence existed for persistent circulation of fetal cells from prior pregnancies. Venous blood samples were obtained from 32 pregnant women and 8 nonpregnant women who had given birth to males 6 months to 27 years earlier. Mononuclear cells were sorted by flow cytometry using antibodies to CD antigens 3, 4, 5, 19, 23, 34, and 38. DNA within sorted cells, amplified by PCR for Y chromosome sequences, was considered predictive of a male fetus or evidence of persistent male fetal cells. In the 32 pregnancies, male DNA was detected in 13 of 19 women carrying a male fetus. In 4 of 13 pregnancies with female fetuses, male DNA was also detected. All of the 4 women had prior pregnancies; 2 of the 4 had prior males and the other 2 had terminations of pregnancy. In 6 of the 8 nonpregnant women, male DNA was detected in CD34+CD38+ cells, even in a woman who had her last son 27 years prior to blood sampling. Our data demonstrate the continued maternal circulation of fetal CD34+ or CD34+CD38+ cells from a prior pregnancy. The prolonged persistence of fetal progenitor cells may represent a human analogue of the microchimerism described in the mouse and may have significance in development of tolerance of the fetus. Pregnancy may thus establish a long-term, low-grade chimeric state in the human female.

Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

The autocrine/paracrine peptide signaling molecules such as growth factors have many promising biologic activities for clinical applications. However, one cannot expect specific therapeutic effects of the factors administered by ordinary drug delivery systems as they have limited target specificity and short half-lives in vivo. To overcome the difficulties in using growth factors as therapeutic agents, we have produced fusion proteins consisting of growth factor moieties and a collagen-binding domain (CBD) derived from Clostridium histolyticum collagenase. The fusion proteins carrying the epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) at the N terminal of CBD (CBEGF/CBFGF) tightly bound to insoluble collagen and stimulated the growth of BALB/c 3T3 fibroblasts as much as the unfused counterparts. CBEGF, when injected subcutaneously into nude mice, remained at the sites of injection for up to 10 days, whereas EGF was not detectable 24 h after injection. Although CBEGF did not exert a growth-promoting effect in vivo, CBFGF, but not bFGF, strongly stimulated the DNA synthesis in stromal cells at 5 days and 7 days after injection. These results indicate that CBD may be used as an anchoring unit to produce fusion proteins nondiffusible and long-lasting in vivo.

Evidence of high expression of peptidylglycine α-amidating monooxygenase in the rat uterus: Estrogen regulation

In the present study, high levels of peptidylglycine α-amidating monooxygenase (PAM), which catalyzes the two-step formation of bioactive α-amidated peptides from their glycine-extended precursors, have been found in the uterus. Expression of PAM was evaluated in the uterus of intact cycling adult female rats and after experimental manipulation of the estrogen status of the rats. During the estrous cycle, PAM mRNA levels exhibited striking changes inversely related to the physiological variations of plasma estrogen levels. The levels of PAM transcripts changed markedly during the estrous cycle, reaching the highest levels at metestrus. There was a 15-fold increase in the abundance of PAM mRNA between metestrus and proestrus. Chronic treatment of ovariectomized rats with 17β-estradiol decreased PAM mRNA levels to values comparable with those found in intact rats at proestrus. Progesterone was without effect on PAM mRNA levels, indicating that the effect was specific for estradiol. In situ hybridization studies were conducted to determine the tissue disposition and cell types expressing PAM. High levels of PAM mRNA were localized in the endometrium at the level of luminal and glandular cells. A weak signal was observed in stromal cells, and the myometrium cells were negative. 17β-Estradiol treatment induced an overall decrease of the hybridization signal, as compared with ovariectomized rats. These results demonstrate the presence of high levels of PAM in the uterus and indicate that estrogens are involved in regulating the expression of the enzyme in this tissue. However, the present study provides no information regarding whether this regulation takes place at the level of transcription or influences mRNA stability.

Chymase cleavage of stem cell factor yields a bioactive, soluble product

Stem cell factor (SCF) is produced by stromal cells as a membrane-bound molecule, which may be proteolytically cleaved at a site close to the membrane to produce a soluble bioactive form. The proteases producing this cleavage are unknown. In this study, we demonstrate that human mast cell chymase, a chymotrypsin-like protease, cleaves SCF at a novel site. Cleavage is at the peptide bond between Phe-158 and Met-159, which are encoded by exon 6 of the SCF gene. This cleavage results in a soluble bioactive product that is 7 amino acids shorter at the C terminus than previously identified soluble SCF. This research shows the identification of a physiologically relevant enzyme that specifically cleaves SCF. Because mast cells express the KIT protein, the receptor for SCF, and respond to SCF by proliferation and degranulation, this observation identifies a possible feedback loop in which chymase released from mast cell secretory granules may solubilize SCF bound to the membrane of surrounding stromal cells. The liberated soluble SCF may in turn stimulate mast cell proliferation and differentiated functions; this loop could contribute to abnormal accumulations of mast cells in the skin and hyperpigmentation at sites of chronic cutaneous inflammation.

Autocrine production and action of IL-3 and granulocyte colony-stimulating factor in chronic myeloid leukemia

Primitive subsets of leukemic cells isolated by using fluorescence-activated cell sorting from patients with newly diagnosed Ph+/BCR–ABL+ chronic myeloid leukemia display an abnormal ability to proliferate in vitro in the absence of added growth factors. We now show from analyses of growth-factor gene expression, protein production, and antibody inhibition studies that this deregulated growth can be explained, at least in part, by a novel differentiation-controlled autocrine mechanism. This mechanism involves the consistent and selective activation of IL-3 and granulocyte colony-stimulating factor (G-CSF) production and a stimulation of STAT5 phosphorylation in CD34+ leukemic cells. When these cells differentiate into CD34− cells in vivo, IL-3 and G-CSF production declines, and the cells concomitantly lose their capacity for autonomous growth in vitro despite their continued expression of BCR–ABL. Based on previous studies of normal cells, excessive exposure of the most primitive chronic myeloid leukemia cells to IL-3 and G-CSF through an autocrine mechanism could explain their paradoxically decreased self-renewal in vitro and slow accumulation in vivo, in spite of an increased cycling activity and selective expansion of later compartments.