Bone Marrow Stromal Cell Layers Research Articles

Abstract 4026: Growth of monoclonal gammopathy of undetermined significance (MGUS) in a 3-dimensional co-culture in vitro model

Abstract Introduction: Multiple Myeloma (MM) is an incurable cancer with a pre-malignant clonal phase of disease called monoclonal gammopathy of undetermined significance (MGUS). Most patients with MGUS do not develop MM and the biology underlying this potential transformation is unclear. Investigations to prevent the development of MM from MGUS is limited by infrequent MGUS progression. MGUS cells have historically proven difficult to grow in vitro because of slow rates of proliferation and difficulty in sustaining cell cultures. An in vitro model of MGUS that circumvents these challenges by recreating a supportive bone marrow microenvironment will allow further investigation into the pathogenesis of disease progression. Herein, we examine a novel stroma-based in vitro model of MGUS and MM using bone marrow stromal cells to support and maintain a bi-directional relationship within the clonal plasma cell population. Aim: To grow MGUS derived plasma cells by recreating a supportive bone marrow microenvironment using a 3-dimensional in vitro model. Methods: We collected a CD38+ cell fraction and a bone marrow stromal fraction from patients with plasma cell dyscrasias using a Miltenyl Biotec column Separator. We seeded stromal cells from a female, kappa expressing MGUS patient or a healthy donor onto a polyglycolic acid/ poly L-lactic acid 90/10 (PLGA) copolymer scaffold. Then, a CD38+ cell fraction from a male patient with lambda secreting PCL or MM cell line U266 or MM1.s (both male, lambda expressing cell lines) were added to create 3D co-culture conditions. For comparison, a 2 dimensional co-culture condition was created by growing MM patient cells or cell lines over a bone marrow stromal cell layer without scaffold.. After one week, samples were formalin fixed and sectioned for immunohistochemistry (IHC) and stained with CD138, kappa, lambda, and ki-67. Fluorescent in situ hybridization (FISH) was used to label X and Y chromosomes. Results: IHC demonstrated proliferation of CD138+ cells evident by Ki-67 expression in in 3-D co-culture systems. There was no growth of the patient derived CD38+ cells in 2-D co-culture and the proliferation of of cell lines was diminished in 2-D versus 3-D. When seeded on healthy donor stroma in 3-D, the CD38+ cells from all sources were lambda restricted. When seeded on the MGUS stroma, the MM1.s cells were lambda restricted while PCL cells showed both kappa and lambda expression with a dominance of kappa expression, and U266 cells showed kappa restriction. FISH demonstrated a female chromosomal pattern with X,Y in the plasma cells from the 3D co-cultures in the case of U266 or PCL cell 3-D co-culture with MGUS stroma. Conclusion: This is the first demonstration of in vitro growth of plasma cells from a patient with MGUS. We speculate the aggressive plasma cells from the U266 cell line and the PCL sample induced the growth of a CD38- cell fraction and caused differentiation to plasma cells. Citation Format: Brea C. Lipe, Omar Aljitawi, Tara Lin, Joseph Fontes. Growth of monoclonal gammopathy of undetermined significance (MGUS) in a 3-dimensional co-culture in vitro model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4026. doi:10.1158/1538-7445.AM2015-4026

Chlorella vulgaris up-modulation of myelossupression induced by lead: The role of stromal cells

In this study, Chlorella vulgaris (CV) was examined for its chelating effects on the ability of bone marrow stromal cell layer to display myeloid progenitor cells in vitro in lead-exposed mice, using the long-term bone marrow culture (LTBMC). In addition, the levels of interleukin (IL)-6, an important hematopoietic stimulator, as well as the numbers of adherent and non-adherent cells were also investigated. Mice were gavage treated daily with a single 50 mg/kg dose of CV for 10 days, concomitant to continuous offering of 1300 ppm lead acetate in drinking water. We found that CV up-modulates the reduced ability of stromal cell layer to display myeloid progenitor cells in vitro in lead-exposed mice and restores both the reduced number of non-adherent cells and the ability of stromal cells from these mice to produce IL-6. Monitoring of lead poisoning demonstrated that CV treatment significantly reduced lead levels in blood and tissues, completely restored the normal hepatic ALA levels, decreased the abnormally high plasma ALA and partly recovered the liver capacity to produce porphyrins. These findings provide evidence for a beneficial use of CV for combination or alternative chelating therapy to protect the host from the damage induced by lead poisoning.

Human embryonic stem cell (hES) derived dendritic cells are functionally normal and are susceptible to HIV-1 infection

BackgroundHuman embryonic stem (hES) cells hold considerable promise for cell replacement and gene therapies. Their remarkable properties of pluripotency, self-renewal, and tractability for genetic modification potentially allows for the production of sizeable quantities of therapeutic cells of the hematopoietic lineage. Dendritic cells (DC) arise from CD34+ hematopoietic progenitor cells (HPCs) and are important in many innate and adaptive immune functions. With respect to HIV-1 infection, DCs play an important role in the efficient capture and transfer of the virus to susceptible cells. With an aim of generating DCs from a renewable source for HIV-1 studies, here we evaluated the capacity of hES cell derived CD34+ cells to give rise to DCs which can support HIV-1 infection.ResultsUndifferentiated hES cells were cultured on S17 mouse bone marrow stromal cell layers to derive CD34+ HPCs which were subsequently grown in specific cytokine differentiation media to promote the development of DCs. The hES derived DCs (hES-DC) were subjected to phenotypic and functional analyses and compared with DCs derived from fetal liver CD34+ HPC (FL-DC). The mature hES-DCs displayed typical DC morphology consisting of veiled stellate cells. The hES-DCs also displayed characteristic phenotypic surface markers CD1a, HLA-DR, B7.1, B7.2, and DC-SIGN. The hES-DCs were found to be capable of antigen uptake and stimulating naïve allogeneic CD4+ T cells in a mixed leukocyte reaction assay. Furthermore, the hES-DCs supported productive HIV-1 viral infection akin to standard DCs.ConclusionPhenotypically normal and functionally competent DCs that support HIV-1 infection can be derived from hES cells. hES-DCs can now be exploited in applied immunology and HIV-1 infection studies. Using gene therapy approaches, it is now possible to generate HIV-1 resistant DCs from anti-HIV gene transduced hES-CD34+ hematopoietic progenitor cells.

Exploratory study of novel leukemia bone marrow stromal cell adhesion mediated drug resistance model

To construct cell adhesion mediated drug resistance (CAM-DR) model based on Acute lymphocyte leukemia bone marrow stromal cells(BMSC) for further studying drug resistance of leukemia. Firstly, we adhesively cultured Jurkat cell strain of human leukaemia lymphocyte with the matrix cell radiated by (60)Co to construct the model of CAM-DR, then evaluated the model in morph and construction by scanning electron microscope. The IC50 of DNR on Jurkat cell was examen by MTT and the concentration and distribution of DNR in the cell was detected by flow cytometry. When the Jurkat cells were cultured with BMSC for 24 h, we found that Jurkat cells had adhesioned the bone marrow stromal cell layer by parapodium and some of them had nichd the mesh consisted by the confluence of BMSC. At the point of 48 h, some Jurkat cells had migrated to underlayer of BMSC, and Jurkat cells were nichd the mesh of BMSC like nidi. The accumulation and distribution of DNR in the Jurkat cells were not affected in the model, but the reaction of Jurkat cells to DNR were significantly inhibited. The model of CAM-DR based on Acute lymphocyte leukemia bone marrow stromal cells was successfully built.

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy

BackgroundMany novel studies and therapies are possible with the use of human embryonic stem cells (hES cells) and their differentiated cell progeny. The hES cell derived CD34 hematopoietic stem cells can be potentially used for many gene therapy applications. Here we evaluated the capacity of hES cell derived CD34 cells to give rise to normal macrophages as a first step towards using these cells in viral infection studies and in developing novel stem cell based gene therapy strategies for AIDS.ResultsUndifferentiated normal and lentiviral vector transduced hES cells were cultured on S17 mouse bone marrow stromal cell layers to derive CD34 hematopoietic progenitor cells. The differentiated CD34 cells isolated from cystic bodies were further cultured in cytokine media to derive macrophages. Phenotypic and functional analyses were carried out to compare these with that of fetal liver CD34 cell derived macrophages. As assessed by FACS analysis, the hES-CD34 cell derived macrophages displayed characteristic cell surface markers CD14, CD4, CCR5, CXCR4, and HLA-DR suggesting a normal phenotype. Tests evaluating phagocytosis, upregulation of the costimulatory molecule B7.1, and cytokine secretion in response to LPS stimulation showed that these macrophages are also functionally normal. When infected with HIV-1, the differentiated macrophages supported productive viral infection. Lentiviral vector transduced hES cells expressing the transgene GFP were evaluated similarly like above. The transgenic hES cells also gave rise to macrophages with normal phenotypic and functional characteristics indicating no vector mediated adverse effects during differentiation.ConclusionPhenotypically normal and functionally competent macrophages could be derived from hES-CD34 cells. Since these cells are susceptible to HIV-1 infection, they provide a uniform source of macrophages for viral infection studies. Based on these results, it is also now feasible to transduce hES-CD34 cells with anti-HIV genes such as inhibitory siRNAs and test their antiviral efficacy in down stream differentiated cells such as macrophages which are among the primary cells that need to be protected against HIV-1 infection. Thus, the potential utility of hES derived CD34 hematopoietic cells for HIV-1 gene therapy can be evaluated.

1046. Derivation of Functionally Normal Macrophages from Human Embryonic Stem (hES) Cells for HIV-1 Gene Therapy

Human embryonic stem cells (hES cells) and their derivatives can be utilized for many promising novel therapies. As a first step towards developing stem cell based gene therapy for AIDS using embryonic stem cells, here we evaluated the capacity of hES cell derived CD34 cells to give rise to normal macrophages. Normal and lentiviral vector transduced hES cells were differentiated into CD34 hematopoietic progenitor cells by culturing on S17 mouse bone marrow stromal cell layers. The hES-CD34 cells were cultured in cytokine media that promotes macrophage differentiation. Our results have shown differentiation of hES-CD34 cells into phenotypically normal macrophages that displayed characteristic cell surface markers CD14, CD4, CCR5, CXCR4, and HLA-DR. Studies evaluating phagocytosis, upregulation of the costimulatory molecule B7.1, and cytokine secretion in response to LPS stimulation suggested that these macrophages are also functionally normal. When challenged with HIV-1, the differentiated macrophages supported productive viral infection. Lentiviral vector transduced hES cells that expressed the transgene GFP also gave rise to macrophages with normal phenotypic and functional characteristics indicating no vector mediated adverse effects during differentiation. These results demonstrated for the first time that functionally normal mature macrophages can be obtained from hES cells and that they can be used in infectious disease research. Now it is possible to test the utility of hES-CD34 cells for anti-HIV gene transduction and evaluation of the differentiated cells such as macrophages for anti- viral resistance in a gene therapy setting. Human embryonic stem cells (hES cells) and their derivatives can be utilized for many promising novel therapies. As a first step towards developing stem cell based gene therapy for AIDS using embryonic stem cells, here we evaluated the capacity of hES cell derived CD34 cells to give rise to normal macrophages. Normal and lentiviral vector transduced hES cells were differentiated into CD34 hematopoietic progenitor cells by culturing on S17 mouse bone marrow stromal cell layers. The hES-CD34 cells were cultured in cytokine media that promotes macrophage differentiation. Our results have shown differentiation of hES-CD34 cells into phenotypically normal macrophages that displayed characteristic cell surface markers CD14, CD4, CCR5, CXCR4, and HLA-DR. Studies evaluating phagocytosis, upregulation of the costimulatory molecule B7.1, and cytokine secretion in response to LPS stimulation suggested that these macrophages are also functionally normal. When challenged with HIV-1, the differentiated macrophages supported productive viral infection. Lentiviral vector transduced hES cells that expressed the transgene GFP also gave rise to macrophages with normal phenotypic and functional characteristics indicating no vector mediated adverse effects during differentiation. These results demonstrated for the first time that functionally normal mature macrophages can be obtained from hES cells and that they can be used in infectious disease research. Now it is possible to test the utility of hES-CD34 cells for anti-HIV gene transduction and evaluation of the differentiated cells such as macrophages for anti- viral resistance in a gene therapy setting.

Transformation of bone marrow B-cell progenitors by E2a-Hlf requires coexpression of Bcl-2.

The chimeric transcription factor E2a-Hlf is an oncoprotein associated with a subset of acute lymphoblastic leukemias of early B-lineage derivation. We employed a retroviral transduction-transplantation approach to evaluate the oncogenic effects of E2a-Hlf on murine B-cell progenitors harvested from adult bone marrow. Expression of E2a-Hlf induced short-lived clusters of primary hematopoietic cells but no long-term growth on preformed bone marrow stromal cell layers comprised of the AC6.21 cell line. Coexpression with Bcl-2, however, resulted in the sustained self-renewal of early preB-I cells that required stromal and interleukin-7 (IL-7) support for growth in vitro. Immortalized cells were unable to induce leukemias after transplantation into nonirradiated syngeneic hosts, unlike the leukemic properties and cytokine independence of preB-I cells transformed by p190(Bcr-Abl) under identical in vitro conditions. However, bone marrow cells expressing E2a-Hlf in combination with Bcl-2, but not E2a-Hlf alone, induced leukemias in irradiated recipients with long latencies, demonstrating both a requirement for suppression of apoptosis and the need for further secondary mutations in leukemia pathogenesis. Coexpression of IL-7 substituted for Bcl-2 to induce the in vitro growth of pre-B cells expressing E2a-Hlf, but leukemic conversion required additional abrogation of undefined stromal requirements and was associated with alterations in the Arf/Mdm2/p53 pathway. Thus, E2a-Hlf enhances the self-renewal of bone marrow B-cell progenitors without inciting a p53 tumor surveillance response or abrogating stromal and cytokine requirements for growth, which are nevertheless abrogated during progression to a leukemogenic phenotype.

Bone marrow stromal cells regulate caspase 3 activity in leukemic cells during chemotherapy

The interaction between leukemic cells and stromal cells of the bone marrow microenvironment has been shown to enhance leukemic cell survival during exposure to chemotherapeutic agents. In the current study we investigated whether association of B lineage acute lymphoblastic leukemic cells with human bone marrow stromal cells altered caspase activation during chemotherapy treatment. Following treatment with Ara-C or VP-16 in vitro, caspase 3 activity in leukemic cells was consistently reduced by co-culture of leukemic cells with human bone marrow stromal cell layers. These observations suggest that the protective effect of the bone marrow microenvironment on leukemic cells may be due, in part, to regulation of caspase 3 activity.

The in-vitro detection and quantitation of ovine bone marrow precursors of multipotential colony-forming cells.

The in-vitro detection and quantitation of ovine bone-marrow precursors of multipotential colony-forming cells (pre-multi CFC) is described. After 5 and 10 days in liquid culture containing medium conditioned by long term bone marrow stromal cell layers (SCM) along with lymph node-conditioned medium (LNCM) or recombinant ovine interleukin-3 (rov IL-3), increased numbers of multi CFC developed from bone marrow precursors as detected by subsequent soft agar clonogenic assay of mixed phenotype colonies. From a variety of cytokines and conditioned media (CM) tested that included recombinant ovine granulocyte-macrophage colony-stimulating factor (rov GM-CSF) and recombinant human macrophage colony-stimulating factor (rhu M-CSF), the combination of SCM plus LNCM or rov IL-3 supported the maximum numbers of multi-CFC in liquid culture. The development of multi CFC from precursors was demonstrated in bone marrow cells treated with a dose of mafosfamide that inactivated > 98% of clonogenic CFC. Quantitative limit dilution analysis of 10 bone marrow samples revealed an average of one pre-multi CFC per 34147 unfractionated cells (range 1:14230-81433). Pre-multi CFC were enriched 38-fold (average) in the 2.0% of bone marrow cells remaining after depletion of lymphocytes and myeloid/erythroid cells expressing the antigen recognized by monoclonal antibody 175. The quantitative assay also revealed preliminary evidence that the pre-multi CFC may consist of subpopulations differing in their sensitivity to mafosfamide.

Establishment of a human pro-B cell line (JKB-1) and its differentiation of preestablished bone marrow stromal cell layer.

A human pro-B cell line, named JKB-1, was established from the bone marrow of a 16-year-old girl with acute lymphoblastic leukemia (ALL) in relapse. The origin of the JKB-1 cell line was indicated by its chromosomal and immunologic similarity to the patient's fresh leukemic cells. This cell line has been growing for more than 14 months in suspension culture medium and had a doubling time of about 24 hours. JKB-1 expressed terminal deoxynucleotide transferase (TdT) and early antigens (HLA-DR, CD19, CD24) of B cells, with heavy chain gene rearrangement. However, it did not express late antigens (CD10, CD20, CD21, CD22, CD23) of B cells, light chain gene rearrangement, and cytoplasmic mu-chain. These results suggested that JKB-1 is at the stage of "pro-B" cell or early B-cell precursors. This cell line was induced to differentiate after 7 days of co-incubation with irradiated bone marrow stromal cells because of the expression of pre-B cell antigens (CD10, CD20), cytoplasmic mu-chain, light chain gene rearrangement, and disappearance of TdT, JKB-1 cells adhered to a preestablished bone marrow stromal cell layer with string-like processes under scanning electron microscope. When JKB-1 cells were separated from the stromal layer by a cyclopore membrane with 0.45 micron pore size, they did not differentiate. Bone marrow stromal cell conditioned medium could not induce differentiation either. Thus it was suspected that direct contact between JKB-1 cells and stromal cells was required for differentiation. In methylcellulose semisolid medium, the colony size and number of JKB-1 cells were increased by stem cell factor (SCF), or interleukin (IL)-3, or IL-7, but they were decreased by IL-6. Moreover, SCF synergized with IL-3 or IL-7 to stimulate the proliferation of JKB-1 cells. Because there are very few reproducible models for examining early stages of human B-cell differentiation, the JKB-1 cell line would be useful for studying the relationship between human B-cell differentiation and bone marrow microenvironment, as well as leukemogenesis.

Carbohydrate-dependent binding of human myeloid leukemia cell lines to neoglycoenzymes, matrix-immobilized neoglycoproteins, and bone marrow stromal cell layers.

The presence of sugar receptors on human myeloid leukemia cells was comparatively assessed by a highly sensitive binding assay, employing a panel of 14 types of neoglycoenzymes (chemically glycosylated Escherichia coli beta-galactosidase). The selected carbohydrate ligands mainly encompass common components of natural glycoconjugates as mono- or disaccharides. The monocytoid cells of the THP-1 line, the very young myeloblasts and the myeloblasts of the lines KG-1a and KG-1, the promyelocytes of the HL-60 line, and the early myeloblasts/erythroblasts of the K-562 line displayed a nonuniform pattern of specific binding with quantitative differences at a fixed, nonsaturating concentration of the probes. Scatchard analysis in four cases corroborated the indication of cell-type-related differences between the various cell lines. To test whether the detectable cellular sugar-binding sites can mediate adhesion to glycoligands, a rather simple model matrix of nitrocellulose-immobilized neoglycoproteins was first used. In comparison to the carbohydrate-free carrier protein significant cell adhesion was observed primarily with neoglycoproteins that exposed galactose, N-acetylgalactosamine, N-acetylglucosamine, mannose, and fucose moieties among the 11 tested types of carbohydrate residue. Subsequently, human bone marrow stromal cell layers were tested as a model matrix with increased levels of physiological relevance and complexity. Mixtures of carbohydrate and neoglycoprotein were employed as inhibitors of an interaction via lectins between the stromal and the tumor cells. The carbohydrate-dependent alterations of this parameter revealed cell-type-associated properties. Tumor cell binding was significantly decreased for not more than two lines with the effective sugars, namely N-acetylgalactosamine, mannose, fucose, and sialic acid.

Measurement of the growth parameters of precursor B-acute lymphoblastic leukaemic cells in co-culture with bone marrow stromal cells; Detection of two CD10 positive populations with different proliferative capacities and survival

A new assay system using the fluorescent probe PKH 26 GL was employed to investigate the regulation of precursor B-cell acute lymphoblastic leukaemic (ALL) cell growth. PKH 26 GL is a lipophilic fluorescent probe which becomes incorporated into the plasma membrane upon the staining of cells. As the amount of probe per cell reduces at each cell division, the fluorescence can be used to measure cell proliferation. Bone marrow derived ALL cells from seven newly diagnosed cases were stained with PKH 26 GL, and cultured for 14 days in control cultures without stimulus, or in cultures with preformed human bone marrow stromal cell layers. Viable leukaemic cells from these cultures were identified on the basis of forward light scatter, 90° light scatter, propidium iodide exclusion, PKH 26 GL staining and CD10 expression by flow cytometry at the beginning of the culture period and on days 2, 6, 10 and 14. The growth parameters of these leukaemic cells were determined by analysis of their pattern of PKH 26 GL fluorescence. A higher rate of proliferation and survival of cells was observed in cultures with stromal cells compared with control cultures, without stromal cells. In the presence of stromal cells, survival and proliferation continued throughout the culture period; in contrast in five of seven control cultures no viable cells could be detected after 6–10 days. Interestingly, two populations of leukaemic cells were distinguished on the basis of their different rates of proliferation, when co-cultured with stromal cells. The results indicate that this technique provides a means for studying and quantitating leukaemic cell growth within a complex stroma-dependent system.

Enrichment and characterization of thymus-repopulating cells in stroma-dependent cultures of rat bone marrow

The bone marrow precursor cells seeding the thymus have been difficult to investigate using fresh bone marrow and in vivo thymus reconstitution assays. We have therefore designed a short-term bone marrow culture system allowing the study of thymus-repopulating cells in the marrow microenvironment. Low-density rat bone marrow cells were grown on pre-established mouse bone marrow stromal cell layers. Cocultured cells were maintained either under steroid-free conditions (Whitlock/Witte-type culture) or in the presence of 10(-7) M hydrocortisone (Dexter-type culture). After 3 days in vitro, the unanchored cell fractions were tested for their ability to colonize and repopulate fetal mouse thymic lobes in vitro. Both fresh low-density cells and Whitlock/Witte-type cultures, but not Dexter-type cultures, gave rise intrathymically to significant numbers of rat donor-type Thy-1.1high CD2+ CD5low CD43+ cells accounting for 50% to 90% of the organ-cultured cells at day 14. Repopulation of fetal mouse thymic lobes by rat Thy-1.1high cells could be used as a readout assay for initiation of thymopoiesis from bone marrow precursor cells, since 90% of the cells were CD3-/low and TCR alpha beta-/low and 15% of the cells co-expressed CD4 and CD8. Dose-response analysis showed that thymus repopulating cells were at least maintained, if not amplified during the 3-day culture period, leading to at least a 10-fold enrichment as compared to unfractionated bone marrow. Unlike fresh low-density cells before culture, short-term Whitlock/Witte-type cultures were depleted in myeloid-restricted precursor cells. In culture, the thymus-repopulating activity was predominantly associated with a 10% lymphoid cell subset which did not express the B-lineage-associated antigens revealed by HIS24 (the rat B220 equivalent) and HIS50 mAbs. We propose that unanchored thymus-repopulating cells enriched in Whitlock/Witte-type cultures may represent lymphoid-restricted, T-cell precursors of the bone marrow capable of emigrating and colonizing the thymus.

Beneficial effects of reduced oxygen tension and perfusion in long-term hematopoietic cultures.

Growth of hematopoietic stem and progenitor cells found in the MNC fraction of human cord blood was evaluated under atmospheres containing reduced (5%) and normal (20%) oxygen tension. Reduced oxygen tension increased total cell numbers by as much as 5-fold in cord blood suspension cultures, but this effect was less pronounced in cultures containing an irradiated bone marrow stromal cell layer. However, reduced oxygen tension resulted in a substantial increase in both the number and frequency of colony-forming cells observed in both types of LTHC studied. Under low oxygen, CFU-C progenitor cell numbers were as much as 10-fold higher. Finally, reduced oxygen tension slowed the rate of irradiated stromal layer degeneration, as judged by cell counts and microscopic examination. These results indicate that low oxygen, which better approximates the in vivo environment, enhances the growth and maintenance of human stromal and progenitor cells in vitro. These low oxygen findings were then applied to a murine model LTHC perfusion system. In this system, irradiated 3T3 stromal layer integrity was improved under low oxygen and was substantially further improved with continuous medium perfusion. Cell counts and flow cytometry analysis indicated that the total cell production and the production of immature cells from murine bone marrow MNC on irradiated 3T3 cells were significantly enhanced under low oxygen with perfusion. After three weeks of culture, a 24-fold higher number of Thy1.2lo F4/80- MAC1- cells (indicative of murine stem and progenitor cells) was observed in the perfusion system as compared with static culture under ambient oxygen.

VH gene family repertoire of resting B cells. Preferential use of D-proximal families early in development may be due to distinct B cell subsets.

The fetal VH gene repertoire was shown previously to be characterized by overrepresentation of D-proximal families, VH 7183 and VH Q52, compared with adult bone marrow B cells in which VH genes were expressed in a more stochastic fashion. To determine the underlying mechanisms of these findings, adult vs fetal progenitors were placed in the same supportive microenvironment and the resulting B lineage cells analyzed for VH gene family expression. The supportive microenvironment was provided by established adult bone marrow stromal cell layers. In this way the relative importance of environmental vs genetic influences could be determined. The fetal B cells and pre-B cells that developed on adult stromal cells maintained a fetal-like VH gene family repertoire with preference for D-proximal families VH 7183 and Q52. In contrast, adult cultured B cells maintained the adult-like repertoire with predominance of the largest family VH J558. Only after long-term incubation was there a change in the expression of particular VH gene families. These findings suggest that the D-proximal VH gene family preference observed early in ontogeny is associated more with the inherent genetic potential of B cell progenitors that predominate during fetal life and less with environmental influences.

Use of gel-well culture chambers as a liquid culture system to measure responses of hematopoietic colony-forming cells to growth factors.

This report presents the results of an investigation in which Gel-Well culture chambers were evaluated for their utility as a liquid culture assay system to measure the responses of hematopoietic colony-forming cells (CFC) to recombinant and cell-derived growth factors. Gel-Wells, designed for anchorage-independent cell growth and diffusion of media components, permitted the weekly replacement of media and growth factors without removing cells from the culture chambers. In these studies, changes in cellularity and CFC content in Gel-Well cultures of human umbilical cord blood cells induced by recombinant interleukin 3 (rIL-3) were quantified. After one week in culture without rIL-3, the number of erythroid burst-forming units (BFU-e) had decreased to 25 +/- 38% of pre-values. In contrast, addition of rIL-3 induced an increase in the number of BFU-e to 390 +/- 135% of pre-values. By three weeks with rIL-3, the number of granulocyte-macrophage colony-forming units (CFU-gm) had increased to 292 +/- 58% of pre-values. Also, the presence of a bone marrow stromal cell layer under the Gel-Well helped to maintain the survival of CFC in liquid culture. These studies demonstrated that Gel-Well culture chambers provide a useful liquid culture system for studying the responses of CFC to growth factors.