Corticosteroid-dependent differentiation of human marrow preadipocytes in vitro

Summary A unique population of human bone marrow-derived, adherent fibroblastlike cells differentiates to adipocyte morphology when grown in vitro in the presence of horse serum and hydrocortisone sodium hemisuccinate. Over the initial 8-weeks growth at 37°C, 7% CO₂, these cells accumulate Oil Red O-positive lipid and form colonies of over 100 cells, which persist in confluent cultures for over 30 weeks. Similar to cultures derived from mouse marrow, corticosteroid-induced adipocyte differentiation is associated with long-term granulopoiesis. Human marrow preadipocytes, as well as human, mouse and rat embryo fibroblast cell lines, failed to differentiate to adipocyte morphology in the presence of insulin. In contrast, the 3T3-L1 insulin-dependent preadipocyte cell line was not induced to differentiate in the presence of hydrocortisone. These studies demonstrate that human marrow preadipocytes are dependent upon corticosteroid for differentiation in vitro. Supported by National Cancer Institute Virus Cancer Program Contract NCI NO1-7-1051.     

Corticosteroid-dependent differentiation of human marrow preadipocytes in vitro

A unique population of human bone marrow-derived, adherent fibroblastlike cells differentiates to adipocyte morphology when grown in vitro in the presence of horse serum and hydrocortisone sodium hemisuccinate. Over the initial 8-weeks growth at 37 degrees C, 7% CO2, these cells accumulate Oil Red O-positive lipid and form colonies of over 100 cells, which persist in confluent cultures for over 30 weeks. Similar to cultures derived from mouse marrow, corticosteroid-induced adipocyte differentiation is associated with long-term granulopoiesis. Human marrow preadipocytes, as well as human, mouse and rat embryo fibroblast cell lines, failed to differentiate to adipocyte morphology in the presence of insulin. In contrast, the 3T3-L1 insulin-dependent preadipocyte cell line was not induced to differentiate in the presence of hydrocortisone. These studies demonstrate that human marrow preadipocytes are dependent upon corticosteroid for differentiation in vitro.     

Immunoregulatory influence of thymic epithelium and thymopoietin on human B-lymphocyte differentiation

Thymic influence upon immunoregulation of B-lymphocyte differentiation in human bone marrow was investigated. Mononuclear cells isolated from marrow of normal adult volunteers were incubated with thymic epithelial monolayers or with the polypeptide thymopoietin. Generation of pokeweed mitogen-stimulated anti-sheep red blood cell antibody-secreting direct plaque-forming cells (PFC) was found to be inhibited following incubation of marrow mononuclear cells with thymic epithelial monolayers. Addition of 50 ng/ml thymopoietin to pokeweed mitogen-stimulated cultures resulted in enhanced marrow PFC responses, whereas higher doses of thymopoietin were inhibitory for the generation of PFC in this assay system. The data suggest that both helper and suppressor T cells are recruited from their precursors in human bone marrow by thymic influences. Generation of helper or suppressor cells may be dependent upon (a) the stage of differentation of precursor T cells and (b) upon the specific action and intensity of the thymic influence.     

Periodically discontinuous induction of bone marrow stem cells toward osteogenic differentiation in vitro

This paper reveals that a discontinuous in vitro induction, namely, the periodic presence and absence of foreign induction factors, might be, under a certain condition, more effective to stimulate stem cells' differentiation than a continuous induction. Bone marrow stem cells (BMSCs) derived from Sprague Dawley rats were employed to examine the effects of discontinuous additions of osteogenic supplements with a series of alternate frequency in contrast to those with continuous induction or no induction. The results demonstrated that a suitable discontinuous induction was more able to achieve osteogenesis than not only no induction but also the associated continuous induction. Additionally, the osteogenic supplements were confirmed to enhance cell differentiation but suppress cell proliferation. So, the combination of differentiation extent per cell and cell number accounts for the "unexpected" good osteogenic effect of the discontinuous induction. The induction effect was found to be dependent upon alternate frequency, and the optimum alternate period in our experimental systems was determined to be around 4 days. Since it is very common to change culture medium every 2-4 days, such a strategy of discontinuous induction does not bring any extra manual work but reduces the consumption of foreign induction factors and significantly enhances the global differentiation efficacy. Our work thus affords a convenient and practical approach to achieve differentiation of BMSCs, which might be useful for potential large-scale culture and differentiation of stem cells. Meanwhile, the existence of optimum frequency implies some unknown inherent rhythms of cell proliferation and differentiation.     

Short in vitro half-life of thymopoietin32--36 pentapeptide in human plasma.

Thymopoietin32--36 (TP5) is a synthetic pentapeptide that has the biological activity of its parent molecule, the 49 amino acid thymic hormone thymopoietin. Tritiated thymopoietin32--36 (3 /-TP5) was prepared by reductive tritiation of dibromotyrosyl-TP5. The stability of 3 H-TP5 in human plasma was studied by analyzing samples by thin-layer chromatography at different time points and quantitating the radioactivity associated with TP5 (Arg-Lys-Asp-Val-Tyr) and its tyrosyl-containing breakdown products (Lys-Asp-Val-Tyr, Asp-Val-Tyr, Val-Tyr, Tyr). In plasma (but not in saline) the pentapeptide was rapidly degraded (apparent t1/2 approximately 30 seconds) with the corresponding appearance of radioactivity associated with the other tyrosyl-containing reference compounds. These data imply that the pentapeptide, which is active in vivo, may rapidly trigger changes in responsive cells; sustained circulating levels may not be required for activity.     

Lamins A and C are not expressed at early stages of human lymphocyte differentiation

Lamins are major proteins of the nuclear envelope that are members of the intermediate filament protein family. In vertebrates, nuclei from differentiated tissues usually contain both lamins of the A and B subtypes, while embryonic tissues contain the B-type lamin only. We have examined the composition of the nuclear lamina in human B and T lymphocytes representative of distinct stages of lymphoid differentiation. We show here that, in both cell lineages, while lamin B is constitutively expressed at all stages of differentiation, A-type lamin expression is restricted to later developmental stages.     

The effect of deoxyguanosine on human lymphocyte function. II. Analysis of the interference with B lymphocyte differentiation in vitro

The differentiation of normal human peripheral blood B lymphocytes into plasma cells in vitro, studied in mononuclear cells stimulated with PWM or in purified B cells stimulated with a T cell-replacing factor (TRF), can be inhibited by both deoxyguanosine (dGuo) and guanosine. The mechanism underlying this effect, which differs from the in vivo findings in PNP deficiency, was analyzed. dGuo toxicity can be antagonized by hypoxanthine but not by deoxycytidine. PNP-deficient and HGPRT-deficient B lymphocytes are not sensitive to the intoxicating properties of (deoxy)guanosine. Inhibition of PNP activity in normal B lymphocytes by 8-aminoguanosine decreases the sensitivity for dGuo intoxication. Incubation of purified B cells (stimulated with TRF) with dGuo leads to increased intracellular levels of guanosine di- and triphosphate (GDP and GTP), whereas deoxyguanosine triphosphate (dGTP) levels remain low. These observations lead to the conclusion that inhibition of B lymphocyte differentiation by dGuo is brought about by one of the end products of the pathway starting with degradation of dGuo by PNP, followed by guanine salvage by HGPRT, and possibly further phosphorylation of GMP into GDP and GTP. According to this mechanism, B lymphocyte differentiation in PNP deficiency is not sensitive to (deoxy)guanosine; because of the absence of PNP activity, these cells cannot accumulate GMP, GDP, and GTP, and therefore escape dGuo intoxication.     

Distinct stages in adipogenesis revealed by retinoid inhibition of differentiation after induction of PPARgamma.

Retinoic acid (RA) inhibits adipocyte differentiation of 3T3-L1 preadipocytes but is effective only early in adipogenesis. RA prevented induction of the adipogenic factors PPARgamma and C/EBPalpha. Using receptor-specific ligands, we determined that the effects of RA were mediated by liganded RA receptors (RARs) rather than retinoid X receptors. Preadipocytes expressed primarily RARalpha and RARgamma; during adipocyte differentiation, RARalpha gene expression was nearly constant, whereas RARgamma1 mRNA and protein levels dramatically decreased. Ectopic expression of RARgamma1 extended the period of effectiveness of RA by 24 to 48h; RARalpha expression had a similar effect, suggesting functional redundancy of RAR subtypes. Remarkably, RA inhibited differentiation when added after PPARgamma1 and PPARgamma2 proteins had already been expressed and resulted in the loss of PPARgamma proteins from cells. By 72 to 96 h after the induction of differentiation, RA failed to prevent differentiation of even ectopic-RAR-expressing cells. Thus, the unresponsiveness of 3T3-L1 preadipocytes to RA after the induction of differentiation is initially due to the reduction in cellular RAR concentration rather than to the induction of PPARgamma. At later times cells continue along the differentiation pathway in a manner which is RA and RAR independent.     

X-ray induction of micronuclei in human lymphocyte subpopulations differentiated by immunoperoxidase staining.

In this study we sought to confirm the radiosensitivity of human peripheral blood lymphocyte subpopulations using a micronucleus assay. Mononucleated cells isolated from peripheral blood were irradiated with X rays. After being cultured for 3 days, cells were fixed and stained using the immunoperoxidase staining technique. Lymphocyte subpopulations were characterized by means of the monoclonal antibodies Leu4 (CD3), Leu2a (CD8) and Leu19 (CD56). Dose-response curves were obtained by scoring the number of micronuclei in binucleated cells that reacted with a specific antibody and were then stained. The dose response of CD8+ (suppressor/cytotoxic) cells was quite similar to that of CD3+ (pan T) cells. In comparison, CD56+ (natural killer) cells were significantly less sensitive, although scorable binucleated CD56+ cells made up less than 4% of the total number of binucleated cells.     

Early stages of testicular differentiation in the rat

Summary The initial phases of the development of the seminiferous cords (future seminiferous tubules) were studied with histological techniques and with electron microscopy. On day 14 after fertilization, seminiferous cords are well differentiated in the anterior part of the testis near the mesonephric tubules. They comprise Sertoli cells which encompass the primordial germ cells. The Sertoli cells show an expanded clear cytoplasm and microfilaments beneath the outer surface; they differentiate complex contact zones. On day 13 a few cells localized near the mesonephric tubules display the characteristics of the Sertoli cells. These cells become more and more numerous. They aggregate and they form the seminiferous cords.The primordia of male gonads explanted in vitro on the mesonephros, realize testicular organogenesis in a synthetic medium. Adding 15% fetal calf serum to the medium prevents the morphogenesis of the testicular cords, although the Sertoli cells seem to differentiate morphologically and physiologically. In these gonads differentiation of the Sertoli cells was obtained but their aggregation and the morphogenesis of the seminiferous cords were prevented. This gives new insights into testicular morphogenesis and probably provides an experimental model for a new type of gonadal anomaly.     

Radiation protection of human lymphocyte chromosomes in vitro by orientin and vicenin.

Orientin (Ot) and Vicenin (Vc), two water-soluble flavonoids isolated from the leaves of Indian holy basil Ocimum sanctum have shown significant protection against radiation lethality and chromosomal aberrations in vivo. In the present study the protective effect of Ot and Vc against radiation induced chromosome damage in cultured human peripheral lymphocytes was determined by micronucleus test. In order to select the most effective drug concentration, fresh whole blood was exposed to 4Gy of cobalt-60 gamma-radiation with or without a 30 min pre-treatment with 6.25, 12.5, 15.0, 17.5 or 20 microM of Ot/Vc. Micronucleus (MN) assay was done by cytochalasin induced cytokinesis block method. Radiation significantly increased the MN frequency (16 times normal). Pre-treatment with either Ot or Vc at all concentrations significantly (P<0.05-0.001) reduced the MN count in a concentration dependent manner, with the optimum effect at 17.5 microM. Therefore, fresh blood samples were incubated with/without 17.5 microM Ot/Vc for 30 min and then exposed to 0.5-4Gy of gamma-radiation. Radiation increased the MN frequency linearly (r(2)=0.99) with dose. Pre-treatment with Ot or Vc significantly (P<0.01-0.001) reduced the MN counts to 51-67% of RT alone values, giving DMFs of 2.62 (Ot) and 2.48 (Vc). Both the compounds showed significant antioxidant activity in vitro at the above concentrations, which was significantly higher than that of DMSO at equimolar concentrations. Thus, the results demonstrate that both the flavonoids give significant protection to the human lymphocytes against the clastogenic effect of radiation at low, non-toxic concentrations. The radioprotection seems to be associated with their antioxidant activity. The clinical potential of these protectors in cancer therapy needs to be investigated.     

Early mouse embryos: Growth and differentiation in vitro

Mouse blastocysts grown in a simple, easily controlled culture for 5 days developed into egg cylinder-like structures. The removal of zona pellucida and treatment with proteolytic enzymes promoted the development of blastocysts in vitro. Egg cylinders similarly cultured grew and showed signs of early organogenesis, i.e., beating heart, blood islands, somites and cephalization.     

In vitro studies on lymphocyte differentiation. II. Generation of terminal deoxynucleotidyl transferase-positive cells in long-term cultures of mouse bone marrow.

The enzyme TdT is the earliest known marker of lymphocytic differentiation in rodents. Cells containing this enzyme were demonstrated in suspension cultures of mouse bone marrow cells that had been maintained in vitro for periods of 7 to 45 days. The cells were detected by immunofluorescence using purified antibodies to homogeneous TdT. Between 0.04 and 2.0% of cultured bone marrow cells from a variety of mouse strains were positive. More than 40% of the TdT-positive cells incorporated 3H-thymidine during a 20-min pulse. Surface Ig and Thy-1 antigens were not detected on the TdT-positive cells. The prevalence of TdT-positive cells was decreased 10-fold in cultures that had been treated with 10(-6) M hydrocortisone 24 hr before harvesting. The results indicate that lymphoid progenitor cells can be generated in vitro.     

In vitro induction of lymphocyte responsiveness by a Strongylus vulgaris-derived mitogen

Proliferation in vitro of peripheral blood lymphocytes both from horses infected with Strongylus vulgaris and from helminth-free ponies was observed in the presence of extracts of the fourth and fifth stage larvae and adults of S. vulgaris. In addition, S. vulgaris extracts induced transformation in cultures of peripheral blood lymphocytes from sheep and dogs and in mouse spleen cell cultures. Nylon wool non-adherent, T cell enriched fractions of lymphocytes from both mice and horses were stimulated by the S. vulgaris larval mitogen while no proliferation was observed in cultures containing nylon wool adherent, B cell enriched fractions. Macrophage co-operation appeared not to be necessary for S. vulgaris mitogen-induced transformation of spleen cells. The S. vulgaris mitogen stimulated a subpopulation of mouse spleen cells different from those responsive to PHA, Con A and LPS. These cells might be T helper cells since B cells were stimulated to proliferate in the presence of both T cells and S. vulgaris larval mitogen. In addition, the supernatant of in vitro cultured larvae of S. vulgaris induced slight, but significant transformation of equine peripheral blood lymphocytes. Therefore, it is possible that the S. vulgaris mitogen released by both viable parasites and degenerating larvae might induce T cell dependent production of immunoglobulin in vivo and account for the beta-globulinaemia, of which IgG(T) is a major component, in S vulgaris infected horses.     

Regulation of lymphocyte proliferation and differentiation by macrophages.

In this paper, we consider the role of a macrophage secretory product in promoting thymocyte differentiation, as well as a macrophage-immune T cell interaction that results in augmented secretion of lymphostimulatory factors. When cultured with the thymocyte-differentiating factor (TDF), thymocytes show a physiological increase in H-2D and K, decreased sensitivity to lysis with anti-TL and complement, and acquisition of responsiveness in the mixed lymphocyte culture. Development of the mature phenotype requires 2 to 3 days of culture and, once attained, is stable. The induced antigenic changes do not require cell division. The activity demonstrated by TDF, which is not attributable to interferon and cannot be replaced by 2-mercaptoethanol, is also displayed by normal thymic macrophages themselves. Enhanced secretion of TDF and of a distinct mitogenic protein follows the interaction of macrophages and immune T cells. This interaction is shown to require physical contact of the two cell types and is regulated by products of the I-A region of the major histocompatibility complex.     

Aryl hydrocarbon hydroxylase induction in human lymphocyte cultures by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity is induced in cultured human lymphocytes by 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin (TCDD) at a concentration in the growth medium 40 to 60 times less than the concentration of 3-methylcholanthrene (MC) necessary for maximal hydroxylase induction. In cultured lymphocytes from 19 individuals, the extent of hydroxylase induction by TCDD or MC ranged between 1.7- and 2.9-fold. Those individuals having (presumably under genetic control) lower basal and MC-inducible hydroxylase activities in their lymphocytes also have lower TCDD-inducible hydroxylase activity. Because of the day-to-day experimental variability, the variations within each assay, and for several other reasons discussed, we suggest that the observed variance of expression of hydroxylase induction more closely fits a unimodal, polygenic ($\text{i.e.}$ 2 or more genes) pattern rather than the trimodal (single gene) form of inheritance proposed recently by Kellermann and coworkers.