Plasma Clot Diffusion Chambers Research Articles

Interferon-gamma-mediated suppression of erythroid progenitor growth by a HLA-DR- and CD4-positive subset of T lymphocytes in acute myeloid leukemia

In this study, we examined the effects of peripheral blood T lymphocytes from patients with acute myeloid leukemia (AML) on marrow-derived erythroid progenitors (BFU-DE and CFU-DE) growth in an in vivo culture by using the plasma clot diffusion chamber (DC) technique. The application of double-compartment chambers (each compartment separated by a membrane filter) makes the investigations of humoral effects of T lymphocytes upon marrow erythroid progenitors proliferation possible. T lymphocytes of AML-patients in the absence of a statistically significant number of monocytes suppressed the growth of BFU-DE and CF-UDE from T lymphocyte- and adherent cell-depleted marrows. The inhibition ability was restricted to the CD4-positive enriched fraction obtained from T cells by using the negative selection technique. In contrast, the CD8-positive enriched fraction had no effect on erythroid colony formation. Autologous and allogeneic BFU-DE and CFU-DE were similarly affected by the CD4-positive T cells. Treatment of T cells with monoclonal antibodies against HLA-DR before cocultures, completely abrogated the suppression of BFU-DE and CFU-DE-derived colony formation. Suppressive activity detected in the CD4-positive T cells was also totally abolished by treatment with anti-interferon-γ antibodies; whereas the inhibition was retained after 30 Gy radiation. Under these experimental conditions, resting T lymphocytes from healthy subjects did not affect the erythroid colony formation. Our data show that in AML-patients, a circulating HLA-DR-positive, less radiosensitive subset within the CD4-positive T cells is capable of inducing an interferon-γ-mediated suppression of erythropoiesis, at least in DC culture.

Diffusion chamber colony‐forming unit (cfu‐d): A primitive stem cell

Assay of hematopoietic precursor cells in diffusion chambers (DCs) implanted intraperitoneally in experimental animals provides a powerful tool for studying stem cell kinetics in vivo. In this system, the effect of cell migration (which complicates whole animal studies) is eliminated because the membranes utilized in the construction of the chambers are impermeable for cells, while permitting free passage of molecules present in the humoral phase of the host. As judged by light microscopy, conditions in the DC cultures primarily favor macrophage and granulocyte growth. However, the use of in vitro and in vivo subculture to further analyze chamber contents has demonstrated that the system supports proliferation of early hematopoietic progenitors. Additionally, cells capable of rescuing lethally irradiated mice proliferate in DC cultures. Development of the plasma clot DC technique has revealed that most of the growth occurs in colonies which are derived from single cells (CFU-d). Characterization of these cells indicates that they are at least as primitive as other colony-forming cells and, also based on subculture studies, can differentiate along several hematopoietic lineages. In addition to normal CFU-d, both embryonal and leukemic cells can give rise to granulocytes, macrophages, megakaryocytes and erythroid cells in the DC cultures. Evaluation of the effects of humoral factors on hematopoietic cell proliferation and differentiation in the system has led to the identification of both stimulators and inhibitors that may be different from the well-characterized cytokines. Thus, the system seems to be useful for detecting molecules controlling the most primitive stages of hematopoiesis. We believe that the DC culture technique holds enormous potential in the study of stem cell proliferation and differentiation in vivo.

Growth of lung cancer in a clonogenic assay using plasma clot diffusion chambers (PCDC) in irradiated mice

Growth of lung cancer in a clonogenic assay using plasma clot diffusion chambers (PCDC) in irradiated mice

An in vivo clonogenic assay for human tumors using plasma clot diffusion chambers implanted in mice

A modification of the in vivo tumor clonogenic assay using plasma clot diffusion chambers has been described which allows improved study of the cytological characteristics of the colonies cultured. Seventy-five percent of the tumors derived from malignant ascites could be cultured successfully (more than 30 small and large colonies per diffusion chamber). The cloning efficiency ranged from 0.01 to 10%. The addition of 2-mercaptoethanol, horse serum and insulin to the diffusion chambers did not affect colony formation, whereas the effect of cell-free malignant ascites added to the diffusion chambers was unpredictable. Colony growth was comparable when fresh or cryopreserved tumor cells were cultured. A linear relationship between the number of tumor cells inoculated and the number of colonies cultured was apparent in the range 10 2–10 4 cells. Colony formation was stimulated by pre-irradiation (8 Gy) of the host animal and by weekly transplantation of the diffusion chambers in new mice. Intravenously administered doxorubicin penetrated the plasma clot and caused inhibition of colony formation in two experiments with melanoma cells.

K 562 cell line in plasma clot diffusion chambers: changes in cell surface phenotype in relationship to culture conditions.

It is well known that proliferation and subsequent differentiation of normal hemopoietic progenitor cells require an appropriate inductive microenvironment and the addition of specific regulatory factors [7]. This is also true for differentiation of leukemic blast cells, which are considered to represent early stages of a hemopoietic cell lineage [6].KeywordsCell Surface PhenotypeGlobin mRNALeukemic Blast CellHuman Chronic Myelogenous LeukemiaHemopoietic Progenitor CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Enhanced acceptance of regenerating bone marrow of random bred newborn mice by random bred adult mice.

Regenerating bone marrow of newborn random bred Sabra mice (9-13 days old) was obtained by the administration of two consecutive i.p. injections of hydroxyurea (HU) (2 x 100 mg/kg body wt), three days prior to collection of the marrow cells. The bone marrow of HU-treated newborn mice was assayed for CFU-S, CFU-C and plasma-clot-diffusion-chamber (PCDC) progenitor cells. A fourfold content of CFU-S was found in the regenerating bone marrow compared with that of the control marrow, while the level of CFU-C and PCDC progenitor cells was the same in treated and untreated newborn mice. In lethally irradiated adult, random bred Sabra recipient mice, transfused with regenerating bone marrow from newborn mice, the initial survival rate was greater than in irradiated animals receiving normal newborn marrow (75% as against 50%); marrow repopulation, 10-14 days after transfusion, was also greater in the former than in the latter group of animals (1.5-2x10(6) nucleated cells per femur as compared with 08.-2x10(5)). The bone marrow of these groups of mice was assayed for CFU-S, CFU-C and PCDC progenitor cells; with a cell inoculum of 5 x 10(4) i.v., 10(5) in vitro and 5 x 10(4) per DC, respectively, pluripotent and committed stem cells were detected in the experimental group and were lacking in control recipients. Regenerating bone marrow of newborn mice was also transfused into lethally irradiated splenectomized recipients. In this experimental group there was high mortality, low marrow repopulation and lack of CFU-S (5-10 x 10(4) cell inoculum). The results of this study indicate that, despite genetic differences among random bred Sabra mice, regenerating bone marrow of newborn mice "takes better" than normal marrow in lethally irradiated recipients. Improved marrow acceptance is possibly due to the increased content of activated CFU-S and/or pre-CFU-S in the regenerating bone marrow

Two Classes of Murine Granulocyte Progenitor Cells Expressed in Plasma Clot Diffusion Chamber Cultures

Two distinct classes of granulocyte progenitor cells present in normal mouse bone marrow are expressed sequentially in the vivo plasma clot diffusion chamber culture system. By several criteria, progenitor cells giving rise to granulocyte colonies on day 4 of culture (CFU-D4) are different from those giving rise to colonies on day 7 (CFU-D7). These differences include: cell cycle activity as measured by in vitro incubation with cytosine arabinoside, residual concentration in the bone marrow after in vivo treatment of donor mice with cytosine arabinoside or methotrexate, resistance to osmotic lysis, size as determined by velocity sedimentation, and the morphology of the granulocyte colonies to which these cells give rise. The CFU-D7 appears to represent an earlier progenitor cell than the CFU-D4 in the differentiation pathway of the granulocyte and is analagous in many respects to the BFU-E in the erythroid pathway.

Two classes of murine granulocyte progenitor cells expressed in plasma clot diffusion chamber cultures

Two distinct classes of granulocyte progenitor cells present in normal mouse bone marrow are expressed sequentially in the vivo plasma clot diffusion chamber culture system. By several criteria, progenitor cells giving rise to granulocyte colonies on day 4 of culture (CFU-D4) are different from those giving rise to colonies on day 7 (CFU-D7). These differences include: cell cycle activity as measured by in vitro incubation with cytosine arabinoside, residual concentration in the bone marrow after in vivo treatment of donor mice with cytosine arabinoside or methotrexate, resistance to osmotic lysis, size as determined by velocity sedimentation, and the morphology of the granulocyte colonies to which these cells give rise. The CFU-D7 appears to represent an earlier progenitor cell than the CFU-D4 in the differentiation pathway of the granulocyte and is analagous in many respects to the BFU-E in the erythroid pathway.

Further Evidence of the In Vivo Role of Erythropoietin or Companion Molecules Induced by Hypoxia on Proliferation and Continuing Differentiation of BFU-e in PCDC

Normal and plethoric bone marrow cells were grown in plasma clot diffusion chambers (PCDC) implanted into the peritoneum of normal mice or mice submitted to 7 her of hypoxia (23,000 ft) daily, on a single day or on 2 consecutive days at different times after implantation of the PCDC's. Daily discontinuous hypoxia (DDH) produced more 6-day bursts than other treatments. Hypoxia on days 1 and 2 after implantation was nearly as effective as DDH on day-6 bursts. Later bouts of hypoxia or a singly hypoxic exposure on day 1 or 2 was less effective. Erythropoietin (Ep) levels were measured by bioassay on both diffusion chamber (DC) contents and serum. Serum Ep levels peaked at 160 mU/ml after a 7-hr hypoxic exposure while the DC content Ep levels were in the nondetectable range (less than 50 mU/ml). The data implies that either higher than normal Ep levels or a companion molecules (s) produced by hypoxia are required for 1-2 days early in the culture period of force an increasing number of BFU-d-e down the erythrocytic pathway and thus increase red cell production at times of need in vivo.

Further evidence of the in vivo role of erythropoietin or companion molecules induced by hypoxia on proliferation and continuing differentiation of BFU-e in PCDC

Normal and plethoric bone marrow cells were grown in plasma clot diffusion chambers (PCDC) implanted into the peritoneum of normal mice or mice submitted to 7 her of hypoxia (23,000 ft) daily, on a single day or on 2 consecutive days at different times after implantation of the PCDC's. Daily discontinuous hypoxia (DDH) produced more 6-day bursts than other treatments. Hypoxia on days 1 and 2 after implantation was nearly as effective as DDH on day-6 bursts. Later bouts of hypoxia or a singly hypoxic exposure on day 1 or 2 was less effective. Erythropoietin (Ep) levels were measured by bioassay on both diffusion chamber (DC) contents and serum. Serum Ep levels peaked at 160 mU/ml after a 7-hr hypoxic exposure while the DC content Ep levels were in the nondetectable range (less than 50 mU/ml). The data implies that either higher than normal Ep levels or a companion molecules (s) produced by hypoxia are required for 1–2 days early in the culture period of force an increasing number of BFU-d-e down the erythrocytic pathway and thus increase red cell production at times of need in vivo.

Growth of human marrow in plasma clot diffusion chambers.

Unseparated as well as nonadherent human bone marrow cells produced colonies while suspended within plasma clots contained within diffusion chambers implanted into irradiated mice. The majority of colonies consisted of granulocytes or macrophages. Colony size was significantly increased by the administration of endotoxin prior to implantation of the chambers.

Assessment of erythrocytic and granulocytic colony formation in an in vivo plasma clot diffusion chamber culture system

Normal rat bone marrow cells seeded into a plasma clot diffusion chamber culture developed into erythrocytic and granulocytic colonies in vivo. Chambers implanted into the peritoneal cavity of normal hosts showed erythrocytic colony numbers reaching an initial peak on day 2, declining on days 3--5, and increasing in a secondary growth phase on day 7. Day 2 colonies were evenly dispersed; day 7 colonies were grouped into discrete areas of bursts. Granulocytic colony numbers reached a peak on day 4 and gradually declined through day 7. Cells in various stages of differentiation could be detected in both colony types. Colony numbers were proportional to the number of marrow cells seeded into the chamber. Host animals treated with phenylhydrazine induced a marked increase in erythrocytic colony numbers and size and a decrease in granulocytic colony formation. Host animals treated with endotoxin suppressed erythrocytic colonies while increasing granulocytic colony size. This method may prove advantageous for the study of hematopoietic colony formation in a physiologic environment.

Assessment of Erythrocytic and Granulocytic Colony Formation in an In Vivo Plasma Clot Diffusion Chamber Culture System

Normal rat bone marrow cells seeded into a plasma clot diffusion chamber culture developed into erythrocytic and granulocytic colonies in vivo. Chambers implanted into the peritoneal cavity of normal hosts showed erythrocytic colony numbers reaching an initial peak on day 2, declining on days 3--5, and increasing in a secondary growth phase on day 7. Day 2 colonies were evenly dispersed; day 7 colonies were grouped into discrete areas of bursts. Granulocytic colony numbers reached a peak on day 4 and gradually declined through day 7. Cells in various stages of differentiation could be detected in both colony types. Colony numbers were proportional to the number of marrow cells seeded into the chamber. Host animals treated with phenylhydrazine induced a marked increase in erythrocytic colony numbers and size and a decrease in granulocytic colony formation. Host animals treated with endotoxin suppressed erythrocytic colonies while increasing granulocytic colony size. This method may prove advantageous for the study of hematopoietic colony formation in a physiologic environment.