Rat islet culture in serum-free medium containing silk protein sericin

Background

The development of islet cultures is desirable for successful clinical islet transplantation. Fetal bovine serum (FBS) has been used as a supplement in islet culture medium, but it may be an unsuitable supplement due recent animal health problems. We have evaluated the use of the silk protein, sericin, derived from Bombyx mori as a replacement for FBS in islet culture medium.

Methods

Twenty rat islets were cultured in medium containing either sericin or FBS, or no supplement, for 14 days, during which time viable islets were counted in order to evaluate islet survival. Insulin secretion was measured in vitro by static incubation on days 3 and 7. In vivo function of cultured islets was tested by syngeneic transplantation. The islets were evaluated histologically and immunohistochemically after culture and transplantation.

Results

Ninety-five percent of islets were viable after culture for 14 days in culture medium supplemented with either FBS or sericin, while no islets survived beyond 7 days in culture without supplement. No significant differences in stimulated insulin secretion were noted between two groups of islets grown on supplemented media. Following transplantation, islets cultured in FBS or sericin rapidly reversed hyperglycemia and maintained normal glycemic control. Histologically, islets cultured with sericin displayed a well-preserved structure and strong insulin staining before and after transplantation.

Conclusion

Serum-free medium containing sericin appears to be useful for islet culture.     

Differentiation of human adipocyte precursors in a chemically defined serum-free medium

Stromal-vascular cells from the inguinal fat tissue of human (age range 1.5 month-27 years), were able to undergo adipose conversion when cultured in a medium containing insulin, transferrin and triiodothyronine. Between 10 and 20 per cent of the cells changed their morphology and accumulated lipid droplets within 10 to 15 days. In most cultures, differentiated cells were present in clusters. These clustered cells were shown by indirect immunofluorescence to contain lipoprotein lipase (located in the Golgi region) and by histochemistry to contain glycerol-3-phosphate dehydrogenase. The occurrence of both enzymes was assessed directly by determining enzyme activities and the synthesis of triacylglycerol was demonstrated by incorporation of [U-14C]glucose into lipids. Foreskin fibroblasts did not display any of these phenotypes. The development of a serum-free, chemically defined medium for the differentiation of diploid adipocyte precursors from human should be of interest for the characterization of factors involved in the stimulation or inhibition of the differentiation process.     

Human marrow mesenchymal stem cell culture: serum-free medium allows better expansion than classical alpha-MEM medium

The expansion of mesenchymal stem cells (MSCs) strongly depends on the culture conditions and requires medium supplemented with 10-20% fetal calf serum (FCS) to generate relevant numbers of cells. However, the presence of FCS is a major obstacle for their clinical use. Therefore, we have evaluated the capacity of expansion of MSC in a commercial serum-free medium (UC) supplemented with a serum substitute (ULTROSER) in comparison with a classical medium alpha-MEM containing 15% FBS. Bone marrow-mononuclear cells collected from 12 volunteer healthy donors were expanded in two different culture media. MSCs isolated in the both media were morphologically similar and expressed identical phenotypic markers. After the primoculture (P0) and one passage, we obtained significantly more MSC and CFU-F progenitors in UC medium than in alphaMEM. Their multipotentiality was preserved during culture, as well as their capacity to support haematopoiesis. In conclusion, our observations strongly suggest that UC is an optimal medium for ex vivo expansion of MSC: it allows a better cell expansion, preserves cell multipotentiality, reduces the culture period and contains low concentration of serum substitute. This medium seems suitable for clinical scale expansion of MSC.     

Quantitation and characterization of human megakaryocyte colony-forming cells using a standardized serum-free agarose assay

Human progenitors of the megakaryocyte (Mk) lineage were detected by their ability to generate colonies containing from 3 to >100 Mk, detectable as glycoprotein IIb/IIIa⁺ cells in APAAP-stained whole mount agarose cultures. Optimal growth conditions were achieved through the use of a defined serum substitute and a suitable cocktail of recombinant cytokines. Under these culture conditions, the smallest Mk-containing colonies (CFC-Mk) were detectable within a week followed by colonies containing larger numbers of Mk over the ensuing 2 weeks. The total number of CFC-Mk at 18–21 d was linearly related to the number of cells plated. Variation in the cytokines added showed that thrombopoietin (TPO) or IL-3 alone would support the formation of large numbers of CFC-Mk. However, optimal yields of colonies containing cells of both Mk and non-Mk lineages required the addition of other growth factors, of which a combination of IL-3, IL-6, GM-CSF and Steel factor (SF) ± TPO was the best of those tested. The further addition of erythropoietin to this combination reduced the number of large ‘pure’ Mk colonies seen and in their place a corresponding number of mixed erythroid-Mk colonies became detectable. Flt3-ligand alone was unable to support the growth of CFC-Mk nor did it enhance their growth when combined with other factors. Plating of FACS-sorted subpopulations of CD34⁺ marrow cells in both serum-free agarose and methylcellulose assays demonstrated that most CFC-Mk are generated from CD34⁺ cells that are CD45RA^? and CD71⁺, approximately half of which are CD41⁺. Thus, CFC-Mk are more similar to primitive clonogenic erythroid progenitors than to their granulopoietic counterparts in their expression of CD34, CD45RA and CD71. Taken together, these findings support the concept that some erythroid and Mk progenitors may share a common developmental pathway. The availability of sensitive and reproducible procedures for isolating and detecting human Mk progenitors should facilitate future investigations of their biology and role in a variety of haematological conditions.     

Wnt signaling promotes hematoendothelial cell development from human embryonic stem cells

Human embryonic stem cells (hESCs) provide an important means to effectively study soluble and cell-bound mediators that regulate development of early blood and endothelial cells in a human model system. Here, several complementary methods are used to demonstrate canonical Wnt signaling is important for development of hESC-derived cells with both hematopoietic and endothelial potential. Analyses using both standard flow cy-tometry, as well the more detailed high-throughput image scanning flow cytometry, characterizes sequential development of distinct early developing CD34(bright)CD31(+)Flk1(+) cells and a later population of CD34(dim)CD45(+) cells. While the CD34(bright)CD31(+)Flk1(+) have a more complex morphology and can develop into both endothelial cells and hematopoietic cells, the CD34(dim)CD45(+) cells have a simpler morphology and give rise to only hematopoietic cells. Treatment with dickkopf1 to inhibit Wnt signaling results in a dramatic decrease in development of cells with hematoendothelial potential. In addition, activation of the canonical Wnt signaling pathway in hESCs by coculture with stromal cells that express Wnt1, but not use of noncanonical Wnt5-expressing stromal cells, results in an accelerated differentiation and higher percentage of CD34(bright)CD31(+)Flk1(+) cells at earlier stages of differentiation. These studies effectively demonstrate the importance of canonical Wnt signaling to mediate development of early hematoendothelial progenitors during human development.     

Responsiveness of glycosyltransferases to thyrotropin in a serum-free culture of porcine thyroid cells

Administration of thyrotropin to porcine thyroid follicles, obtained in a serum-free chemically defined medium, provoked marked increases in the activities of several glycosyltransferases involved in protein N-glycosylation. The coincidence of these effects with a previously demonstrated enhancement of thyroglobulin production renders a relationship between these events likely. The most important stimulation was for peptide oligosaccharyltransferase (3-fold). Among the enzymes involved in the synthesis of the lipid oligosaccharide donor, Dol-P glycosyl- and mannosyltransferases were increased 1.5-fold, and Dol-P N-acetylglucosaminylphosphotransferase only 1.15-fold. As regards terminal glycosyltransferases, asialofetuin sialyltransferase was increased 2-fold and ovomucoid galactosyltransferase only 1.2-fold. There was a continuous release of the latter two enzymes into the culture medium.     

Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions

Chemically defined medium (CDM) conditions for controlling human embryonic stem cell (hESC) fate will not only facilitate the practical application of hESCs in research and therapy but also provide an excellent system for studying the molecular mechanisms underlying self-renewal and differentiation, without the multiple unknown and variable factors associated with feeder cells and serum. Here we report a simple CDM that supports efficient self-renewal of hESCs grown on a Matrigel-coated surface over multiple passages. Expanded hESCs under such conditions maintain expression of multiple hESC-specific markers, retain the characteristic hESC morphology, possess a normal karyotype in vitro, as well as develop teratomas in vivo. Additionally, several growth factors were found to selectively induce monolayer differentiation of hESC cultures toward neural, definitive endoderm/pancreatic and early cardiac muscle cells, respectively, in our CDM conditions. Therefore, this CDM condition provides a basic platform for further characterization of hESC self-renewal and directed differentiation, as well as the development of novel therapies.     

Thrombopoietin alone stimulates the early proliferation and survival of human erythroid, myeloid and multipotential progenitors in serum-free culture

We examined the effects of recombinant human thrombopoietin (TPO, c-Mpl ligand) on the proliferation and differentiation of human haemopoietic progenitors other than megakaryocytic progenitors using serum-free cultures. TPO alone supported the generation of not only megakaryocytic (MK) but also blast cell (blast) colonies from cord blood CD34⁺ cells. Delayed addition of a cytokine cocktail (cytokines; interleukin (IL)-3, IL-6, stem cell factor, erythropoietin, granulocyte-macrophage colony-stimulating factor, and TPO) to cultures with TPO alone on day 7 induced various colonies including granulocyte-macrophage (GM) colonies, erythroid bursts (E), granulocyte-erythrocyte-macrophage-megakaryocyte (GEMM) colonies. Replating experiments of blast colonies supported by TPO alone for culture with cytokines revealed that approximately 60% of the blast colonies contained various haemopoietic progenitors. Single cell cultures of clone-sorted CD34⁺ cells indicated that TPO supported the early proliferation and/or survival of both primitive and committed haemopoietic progenitors. In serum-free suspension cultures, TPO alone significantly stimulated the production of progenitors for MK, GM, E and GEMM colonies as well as long-term culture-initiating cells. These effects were completely abrogated by anti-TPO antibody. These results suggest that TPO is an important cytokine in the early proliferation of human primitive as well as committed haemopoietic progenitors, and in the ex vivo manipulation of human haemopoietic progenitors.     

Growth of normal human glial cells in a defined medium containing platelet-derived growth factor.

DNA synthesis and cell division were measured in serum-free cultures of human normal diploid glial cells maintained in MCDB 105 medium. In growth factor-free cultures the cells remained viable but the cell number was essentially constant. Supplementation with 10 ng of epidermal growth factor or platelet-derived growth factor per ml significantly stimulated DNA synthesis and cell multiplication. Growth occurred both when cells were allowed to settle in serum-containing medium and when cells were plated in serum-free medium. In the latter type of cultures, the cell yield was improved bu incubating the cells in collagen-coated dishes. The use of a miniclone technique allowed the analysis of cell multiplication induced by platelet-derived growth factor at the clonal level and demonstrated that the growth factor induced several cell cycle rounds in a large fraction of clones. The results show that normal cells grown in a recently developed synthetic medium (MCDB 105) supplemented with pure growth factors may multiply without the addition of plasma-derived factors ("progression factors"). It is suggested that the need fo progression factors may simply depend on the composition of the synthetic nutrient medium.     

人胚胎干细胞研究展望

一、 胚胎干细胞(embryonic stem cell,ES细胞)的获取途径及其特征Thomson实验室利用合法获得的14个体外受精形成的囊胚中的内细胞群,以抗BeWO细胞的单克隆抗体作免疫标记,用免疫切除法去除滋养层,以γ射线照射的小鼠胚胎成纤维     

表皮细胞培养条件的优化

目的探讨生长因子（EGF）在人表皮细胞无血清培养中的作用,寻找较好组合．以期加快表皮细胞成皮。方法选择EGF、胰岛素、霍乱毒素、氢化考的松4个实验因素,每个因素设立4个浓度水平。利用L16（4X4）正交表设计试验,考察不同培养条件对表皮细胞生长情况、克隆形成率的影响。结果EGF在无血清培养中对培养人表皮细胞的生长及增殖有明显促进作用（P〈0．01）,在一定范围内（5～20ng／m1）随EGF浓度提高,效应加强;胰岛素浓度在5g／ml时对培养细胞的生长及增殖影响最大;霍乱毒素浓度在4．2ng／ml时作用最好;氢化考的松作用不明显。EGF、胰岛素、霍乱毒素、氢化考的松分别取20ng／ml、5μg／ml、8．4ng／ml、0．2μg／ml浓度时细胞生长及增殖效果最好。结论采用此优化的培养方法,表皮细胞的生长比较迅速,分化不太明显,可保持较旺盛的增殖力。     

X-inactivation in female human embryonic stem cells is in a nonrandom pattern and prone to epigenetic alterations

X chromosome inactivation (XCI) is an essential mechanism for dosage compensation of X-linked genes in female cells. We report that subcultures from lines of female human embryonic stem cells (hESCs) exhibit variation (0-100%) for XCI markers, including XIST RNA expression and enrichment of histone H3 lysine 27 trimethylation (H3K27me3) on the inactive X chromosome (Xi). Surprisingly, regardless of the presence or absence of XCI markers in different cultures, all female hESCs we examined (H7, H9, and HSF6 cells) exhibit a monoallelic expression pattern for a majority of X-linked genes. Our results suggest that these established female hESCs have already completed XCI during the process of derivation and/or propagation, and the XCI pattern of lines we investigated is already not random. Moreover, XIST gene expression in subsets of cultured female hESCs is unstable and subject to stable epigenetic silencing by DNA methylation. In the absence of XIST expression, approximately 12% of X-linked promoter CpG islands become hypomethylated and a portion of X-linked alleles on the Xi are reactivated. Because alterations in dosage compensation of X-linked genes could impair somatic cell function, we propose that XCI status should be routinely checked in subcultures of female hESCs, with cultures displaying XCI markers better suited for use in regenerative medicine.     

Effects of insulin and insulin-like growth factor I on growth of human leukemia cells in serum-free and protein-free medium

Human myeloid leukemia cells (HL60) and malignant lymphocytes (Namalwa) were grown in protein-free, Fe-supplemented media and used to study growth responses to insulin and insulin-like growth factor 1 (IGF-I). HL60 cells previously grown in serum-free medium containing microgram quantities of insulin showed an 18-fold reduction in cumulative cell production when grown without insulin. However, the same cells showed reduced or absent growth stimulation with 1 to 100 ng/mL insulin or IGF- I for at least four days following insulin deprivation, indicating that culture conditions modified insulin and IGF-I responses. When the same cells were grown in Fe-supplemented, protein-free medium (RPMI-Fe), insulin and IGF-I caused dose-dependent stimulation of HL60 cell growth with half-maximal stimulation at nanogram concentrations. Namalwa cells grown in protein-free medium showed no response to either hormone. Radioligand binding showed the presence of insulin and IGF-I receptors on both HL60 and Namalwa cells grown in RPMI-Fe. HL60 cells grown in fetal bovine serum had higher, and cells grown with microgram quantities of insulin dramatically reduced, insulin binding. Competitive binding studies and cultures with anti-IGF-I receptor antibody showed insulin and IGF-I stimulated growth through their respective specific receptors. Both insulin and IGF-I stimulate growth of some cultured human leukemia cells, but the presence of insulin or IGF-I receptors alone does not predict growth responses. Culture conditions affect both cellular responses and ligand binding by these hormones and must be closely controlled to study growth responses.     

Hormonal control of human colon carcinoma cell growth in serum-free medium.

A human colon carcinoma cell line, HC84S, was established in serum-supplemented medium from a colon tumor line T84 transplanted in nude mice. These cells also grew in a serum-free, synthetic medium supplement with insulin, glucagon, epidermal growth factor, transferrin, hydrocortisone, triiodothyronine, selenium, and ascorbic acid. HC84S cells grew 3 times faster in this medium than in serum-containing medium and formed gland-like structures closely resembling the original tumor morphologically. In serum-containing medium, the cells grew as a monolayer and did not form such structures. Primary cultures from transplantable human colon tumor lines maintained in nude mice and a primary tumor from a patient were established directly in this hormone-supplemented medium in collagen-treated plastic dishes without fibroblast overgrowth. The hormone-supplemented medium may be generally useful for the establishment of human colon carcinoma cell lines.     

Proliferation and differentiation of myelodysplastic CD34+ cells in serum-free medium: II. Response to combined colony-stimulating factors

Abstract: To investigate the role of colony stimulating factors (CSFs) in the proliferation and differentiation of progenitor cells from myelodysplastic syndromes (MDS), marrow progenitor cells from 18 MDS patients were highly purified using CD34 monoclonal antibody and immunomagnetic microspheres (MDS CD34+ cells). These cells were cultured in serum-free medium with various combinations of five colony stimulating factors (CSFs): recombinant human interleukin-3 (rIL-3), granulocyte/macrophage-CSF (rGM-CSF), granulocyte-CSF (rG-CSF), macrophage-CSF (rM-CSF), and erythropoietin (rEP). Among the tested CSFs, such as rM-CSF, rG-CSF, rGM-CSF and rIL-3, a combination of the first three CSFs was the most effective stimulus for the proliferation of non-erythroid MDS progenitor cells. An increase of undifferentiated “blast” cell colonies in 5/18 MDS patients occurred and these 5 patients belonged to the high-risk group. In the presence of these three CSFs, rIL-3 had no effect on the proliferation and differentiation of MDS CD34+ cells; however, IL-3 was efficient for the proliferation of MDS CD34+ cells to the erythroid lineage. rGM-CSF or rIL-3 alone did not efficiently support proliferation and differentiation of CD34+ cells. M-CSF is present in normal human serum at a concentration of 550 ±110 U/ml, a concentration exceeding that used in this study (100 U/ml). Therefore, in vivo administration of G-CSF combined with GM-CSF to MDS patients may be one of the most effective CSF combinations for proliferation of MDS progenitor cells to the non-erythroid lineage. However, the effect on the capacity for differentiation was minimal, especially in patients belonging to the high-risk group.