Importance of serum source for the in vitro replicative senescence of human bone marrow derived mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) may be used for therapeutic applications. Culture conditions such as the serum source may impact on cell quality and the onset of replicative senescence. We have examined the effect of culturing hMSCs in autologous serum (AS) versus fetal bovine serum (FBS) on factors involved in in vitro replicative senescence. hMSCs from four donors were cultured in 10% FBS or 10% AS until they reached senescence. Cells were harvested at early passage and near senescence to study factors known to be involved in cellular senescence. The number of population doublings till senescence was similar for cells cultured in FBS, but varied greatly for hMSCs cultured in AS. FBS cells accumulated in S phase of cell cycle. This could not be explained by increased expression of cell cycle inhibitor proteins. Heat shock proteins were upregulated in AS compared to FBS cells. Reactive oxygen species and nitric oxide were upregulated in senescent FBS cells. Telomeres were shorter in senescent cells, more significantly in FBS cells. The source of serum was a determinant for the time till senescence in cultured hMSC. Serum source affected aspects of cell cycle regulation and the levels of heat shock proteins. Several mechanisms are likely to be responsible for replicative senescence in hMSC. Insight into the molecular details of how serum factors impacts on these mechanisms is important for the safe use of hMSCs in clinical applications.     

Inhibitory effects of human serum on human fetal skin fibroblast migration: Migration-inhibitory activity and substances in serum, and its age-related changes

Summary In order to clarify the environmental factors modulating cell migration, we investigated the effects of human serum on cell migration, and found that serum from adult donors strongly (by 48%) suppressed the migration of human fetal skin fibroblasts into a denuded area in a cell monolayer. Human serum from old donors inhibited cell migration more strongly than that from adult donors. Next, we investigated the properties of migration-inhibitory activity of human serum and serum proteins in order to identify migration-inhibitory substances. Human serum from adult donors strongly suppressed the migration of human fetal skin fibroblasts, although it stimulated cell proliferation more strongly than fetal bovine serum (FBS), indicating that the inhibitory effects of human serum on cell migration was not due to its toxic effects. The inhibition of cell migration by human serum was concentration dependent. It was demonsstrated that the inhibition did not depend on the inhibitory effects of human serum on collagen synthesis. The migration-inhibitory activity was seen in fractions over 100 kDa, as determined by an ultrafiltration membrane, and no inhibitory activity was observed in fractions under 100 kDa. On the other hand, it was not detected either in fractions over 100 kDa or under 100 kDa in FBS. Among the over 100 kDa human serum proteins examined, γ-globulin, α2-macroglobulin, and low density lipoprotein (LDL) suppressed fibroblast migration in a concentration-dependent manner. However, among the three, cell migration-inhibiting activity of γ-globulin almost disappeared when cell migration was conducted in 10% FBS-supplemented medium. These results indicated that α2-macroglobulin and LDL were candidate substances for cell migration-inhibiting activity in human serum.     

Reduced frequency of sister chromatid exchanges in human lymphocytes cultured with autologous serum

Summary The incidence of sister chromatid exchange (SCE) was determined in human lymphocytes cultured with fetal calf, human AB, and autologous serum. In each individual studied, cells grown in medium supplemented with fetal calf and human AB serums showed higher yields of SCE than those cultured with autologous serum. Increased concentration of fetal calf and human AB serum in the tissue culture medium resulted in elevated frequency of SCE. No such elevation in SCE frequency was observed with increased concentration of autologous serum. The results indicate the presence of extraneous SCE-inducing factors in fetal calf and human AB serum, the nature of which is not precisely known.Aided by C.S.I.R. Grant No. 7/45 (1052/77) EMR I     

Role of human serum and cellular factors on the growth of peripheral blood CFU-gm

The effects of fetal calf serum (FCS), autologous serum (AS) and pooled human serum (PS) on granulocyte-macrophage progenitors (CFU-gm) from normal human peripheral blood mononuclear non-adherent cells (MNAC) were studied to determine optimal growth conditions. PS provided fewer variations and better growth conditions than AS and FCS. Moreover, serum inhibitors affecting both autologous and heterologous CFU-gm were detected in a small group (10%) of donors. Since CFU-gm from different donors display a relatively uniform cell-cycle status and membrane phenotype, under optimal growth conditions the number of CFU-gm probably reflects the size of the circulating myeloid progenitor cell compartment.     

Effectiveness of autologous serum as an alternative to fetal bovine serum in adipose-derived stem cell engineering

In cell culture, medium supplemented with fetal bovine serum is commonly used, and it is widely known that fetal bovine serum supplies an adequate environment for culture and differentiation of stem cells. Nevertheless, the use of xenogeneic serum can cause several problems. We compared the effects of four different concentrations of autologous serum (1, 2, 5, and 10 %) on expansion and adipogenic differentiation of adipose-derived stem cells using 10 % fetal bovine serum as a control. The stem cells were grafted on nude mice and the in vivo differentiation capacity was evaluated. The isolation of adipose-derived stem cells was successful irrespective of the culture medium. The proliferation potential was statistically significant at passage 2, as follows: 10 % autologous serum >10 % fetal bovine serum = 5 % autologous serum >2 % autologous serum = 1 % autologous serum. The differentiation capacity appeared statistically significant at passage 4, as follows: 10 % fetal bovine serum >10 % autologous serum = 5 % autologous serum >2 % autologous serum = 1 % autologous serum. Ten percent autologous serum and 10 % fetal bovine serum had greater differentiation capacity than 1 and 2 % autologous serum in vivo, and no significant difference was observed between the groups at ≥5 % concentration at 14 weeks. In conclusion, 10 % autologous serum was at least as effective as 10 % fetal bovine serum with respect to the number of adipose-derived stem cells at the end of both isolation and expansion, whereas 1 and 2 % autologous serum was inferior.     

Effect of serum on cells cultured from human mammary tumors.

Clusters of cells derived from biopsy specimens of human mammary ductal carcinomas form two morphologically distinct epithelial colonies in culture, designated as E and E'. The proportion of E' cell clusters that attached and formed colonies ranged from 0.3 to 13.0% with different tumors. Attachment was independent of tumor grade. Microscopic observations revealed that the survival of E' cell colonies was limited to approximately 10 days with rapid cell degeneration commencing about 7 days. A comparison of sera showed that colony formation by cells from malignant tumors during the 1st week of culture was maximum in the presence of fetal bovine serum. Human serum alone was 70 to 100% less effective in promoting E' colonies. The most significant finding was that human serum from normal donors inhibited E' colony development in the presence of FBS. Although human serum was less effective than FBS in promoting colony formation by clusters of E cells, an inhibition was not observed. Inhibitory activity could not be attributed to either antagonistic hormones or the source of human serum. These results demonstrate that normal human serum contains a factor(s) that exhibits an inhibitory activity specific for human epithelial cells (E') derived from malignant tumors.     

Effect of serum on prostaglandin production during the co-culture of human thyroid cells and peripheral blood mononuclear cells

Prostaglandin generation by human peripheral blood mononuclear cells is enhanced during co-culture with human thyroid cells. The objective of the present study was to determine the influence of various sera on this process. Human thyroid adenoma cell monolayers were cultured with normal human peripheral blood mononuclear cells for three days in the presence of a variety of sera, or serum fractions. Prostaglandin E (PGE) in the medium was measured by bioassay or by radioimmunoassay. Significantly more PGE was generated in cultures containing fetal calf serum than in those containing human serum. This difference was not abolished by dialysis of the human serum. When the 50% (NH₄) ₂SO₄ precipitate of the serum was used, PGE generation was similar to that in fetal calf serum, indicating the presence of an inhibitory factor in human serum. The degree of this inhibitory activity was similar in autologous and heterologous human serum, as well as in normal subjects and patients with Graves' disease. Gel filtration and ion-exchange chomatography of human serum showed the inhibitor to co-migrate with albumin. Evidence presented suggests that the inhibitor is not albumin itself but is, instead, a factor tightly bound to albumin. Inhibitory activity was also found in rabbit, goat, rat and cow serum. Prostaglandins are potent modulators of immune-cell function. These data indicate that this process may be modulated by a factor in mammalian serum. The relative absence of this factor in fetal serum may have important implications in regard to the profound changes which occur in the immune system after birth.     

Effect of serum on cells cultured from human mammary tumors

Summary Clusters of cells derived from biopsy specimens of human mammary ductal carcinomas form two morphologically distinct epithelial colonies in culture, designated as E and E′. The proportion of E′ cell clusters that attached and formed colonies ranged from 0.3 to 13.0% with different tumors. Attachment was independent of tumor grade. Microscopic observations revealed that the survival of E′ cell colonies was limited to approximately 10 days with rapid cell degeneration commencing about 7 days. A comparison of sera showed that colony formation by cells from malignant tumors during the 1st week of culture was maximum in the presence of fetal bovine serum. Human serum alone was 70 to 100% less effective in promoting E′ colonies. The most significant finding was that human serum from normal donors inhibited E′ colony development in the presence of FBS. Although human serum was less effective than FBS in promoting colony formation by clusters of E cells, an inhibition was not observed. Inhibitory activity could not be attributed to either antagonistic hormones or the source of human serum. THese results demonstrate that normal human serum contains a factor(s) that exhibits an inhibitory activity specific for human epithelial cells (E′) derived from malignant tumors. Supported by NCI Contract CB-33898 and a Fellowship from the Imperial Cancer Research Fund, London, England.     

Rat marrow-derived mesenchymal stem cells developed in a medium supplemented with the autologous versus bovine serum

The controversial effect of autologous serum (AS) on human mesenchymal stem cells (MSC) was studied in rat MSC culture. Rat bone marrow cells were plated in a medium containing either FBS (fetal bovine serum) or AS were cultured to passage 3, during which the population doubling number (PDN) of both cultures were measured and compared statistically. The number of viable cells, the cell colonogic activity, and cell growth rate were also compared. In addition, mineralization in the osteogenic cultures from each system was measured. Our data indicated that AS enriched medium provided a microenvironment in which growth rate as well as bone differentiation of the isolated MSCs were significantly higher than in FBS enriched medium.     

Human autologous serum obtained using a completely closed bag system as a substitute for foetal calf serum in human mesenchymal stem cell cultures

The major problem in cell therapy is the possibility of viral or bacterial infection and immune reactions. Therefore, it is expected of culture cells which are intended to be re-implanted with autologous serum rather than conventional bovine serum. Cell therapy with human mesenchymal stem cells (hMSC), differentiating to various cells, is thought to be curative. To culture hMSC with human autologous serum (HAS) and re-implant them for cell therapy, we developed a completely closed bag system separating serum, comparing proliferation and multipotency of hMSC cultured in HAS with those in foetal calf serum (FCS). HAS was simply, safely and efficiently obtained with the developed closed bag system. Cell proliferation of hMSC cultured in HAS was greater than that in FCS. hMSC, exposed to the defined induction medium containing HAS as well as FCS, differentiated into osteoblasts and adipocytes. These findings suggest that HAS obtained with the developed closed bag system is advantageous in a point of decrease in risk of virus or bacterial infection and foreign protein contamination and enhancement of proliferation of hMSC.     

Towards xeno-free cultures of human limbal stem cells for ocular surface reconstruction

Isolated limbal epithelial stem cells (LESCs) were cultured with or without a 3T3 murine fibroblast feeder-layer (FL) in 4 different culture media on culture plates or on denuded human amniotic membrane (AM) support and fibrin gel support: (1) control medium supplemented with fetal bovine serum; (2) control medium supplemented with the synthetic serum “XerumFree? XF205” (XF); (3) CnT-20 medium supplemented with “XerumFree? XF205” (CnT-XF) and (4) CnT-20 medium supplemented with human AB serum (CnT-AB). The three xenogeneic media were compared to standard condition (control + FL) and parameters assessed included cell morphology, proliferative potential, number of passages, assessment of clonogenic and abortive colonies, life span, ?Np63α expression and epithelial morphology on AM. During serial cultivation of LESCs, most of the tested xeno-free media supported similar numbers of cell passages, total colony number, cumulative cell doublings (CCD) rates and expression of ?Np63α compared to control. The conditions cultivated with a FL showed a non-statistically significant higher number of cell passages and CCD rates before senescence when compared to the same conditions cultured without FL. Except for the control medium, only XF medium enabled the growth of cells on AM. The expression of ?Np63α was comparable in all the cultures grown onto AM, when compared to the controls on fibrin gel. In conclusion, the xeno-free media enabled LESC culture both on plastic and on denuded human AM. Despite the analyses were carried out in a statistically low number of samples and need re-assessment in a larger cohort, our results suggest that the production of a completely xeno-free LESC graft could be beneficial for future clinical applications.     

Heparin inhibits SMC growth in the presence of human and fetal bovine serum

Heparin (HP) has antiproliferative as well as anticoagulant properties, but not all HP preparations are equally antiproliferative. A recent report found that HP lost its total antiproliferative activity when fetal bovine serum (FBS) was replaced with human serum (HS) in culture media. This observation led to the investigation of our most potent antiproliferative Upjohn HP preparation effects on bovine pulmonary artery smooth muscle cells (PASMC) and systemic SMC growth stimulated in the presence of either FBS or HS. Bovine PASMC, human PASMC, and bovine aortic SMC were treated with 10 microg/ml Upjohn HP in either 15% FBS or 15% HS and the cell number was determined by a Coulter counter. We found that Upjohn HP significantly inhibited bovine PASMC and systemic SMC proliferation in both HS and FBS. The antiproliferative activity of the above HP preparation in HS may lead to an effective treatment of pulmonary vascular and systemic remodeling.     

Differentiation of lymphoid cells. A selective anti-mitogenic component of normal serum which inhibits the induction of immunoglobulin production.

Rabbit lymph node cell populations cultured in vitro in the presence of fetal calf serum are induced to produce immunoglobulin M-secreting cells. The induction of such immunoglobulin production, measured by the capacity of the cell population to secrete immunoglobulins, was inhibited when cells were cultured with sera from a variety of species despite the presence of fetal calf serum. The addition of such inhibitory serum 36 hours after initiation of the cell culture or thereafter was without effect on the extent of induction of immunoglobulin production. On the other hand, the presence of inhibitory serum in culture during only the first 24 hours yielded the same inhibition as when serum was present throughout the 72-hour culture period. Inhibitory sera also suppressed the incorporation of thymidine into DNA. The induction of immunoglobulin production and the incorporation of thymidine into DNA were essentially equally inhibited by the same range of serum concentrations. Unlike conventional inhibitors of DNA synthesis, the inhibitory sera exhibited selective specificity with regard to the kind of cells that could be affected. Thus, such sera inhibited the DNA synthesis of lymph node cells cultured in the presence of fetal calf serum but did not inhibit concanavalin A-stimulated DNA synthesis of such cultured cells and, similarly, serum did not inhibit DNA synthesis of thymus cells cultured in the presence of fetal calf serum. The sera of all species examined were inhibitory except for fetal sera. As judged from a quantitative assay, bovine and porcine serum contained the highest titer of inhibitor, whereas sera from human, rat, mouse, and rabbit were clustered in a group exhibiting less inhibitor. Ascites fluid and lymph node extracellular fluids contained less inhibitor than found in the serum of the same animal and lysates of washed lymph node cells were devoid of inhibitor. Although fetal bovine serum and newborn bovine serum did not contain the inhibitor, it was detectable within 24 hours of parturition. The inhibitor is of relatively large apparent molecular weight (about 300,000) and has been purified about 70-fold.     

Forskolin-loaded human serum albumin nanoparticles and its biological importance

Abstract

In this study, forskolin-loaded human serum albumin nanoparticles (FR-HSANPs) were successfully prepared by incorporation and affinity-binding methods. FR-HSANPs were characterized by transmission electron microscope that most of them are circular in shape and size is around 340?nm. The drug loading was more than 88% and further sustained release profiles were observed as it is 77.5% in 24?h time. Additionally, the cytotoxicity results with HepG2 cells indicated that FR-HSANPs showed significantly higher cytotoxicity and lower cell viability as compared to free forskolin (FR). Furthermore, to understand the binding mechanism of human serum albumin (HSA) with forskolin resulted from fluorescence quenching as a static mechanism and the binding constant is 6.26?±?0.1?×?10⁴ M^?1, indicating a strong binding affinity. Further, association and dissociation kinetics of forskolin–HSA was calculated from surface plasmon resonance spectroscopy and the binding constant found to be K_forskolin = 3.4?±?0.24?×?10⁴ M^?1 and also fast dissociation was observed. Further, we used circular dichroism and molecular dynamics simulations to elucidate the possible structural changes including local conformational changes and rigidity of the residues of both HSA and HSA–forskolin complexes.

Communicated by Ramaswamy H. Sarma     

Genetic and epigenetic instability of human bone marrow mesenchymal stem cells expanded in autologous serum or fetal bovine serum

Culture of mesenchymal stem cells (MSCs) under conditions promoting proliferation and differentiation, while supporting genomic and epigenetic stability, is essential for therapeutic use. We report here the extent of genome-wide DNA gains and losses and of DNA methylation instability on 170 cancer-related promoters in bone marrow (BM) MSCs during culture to late passage in medium containing fetal bovine serum (FBS) or autologous serum (AS). Comparative genomic hybridization indicates that expansion of BMMSCs elicits primarily telomeric deletions in a subpopulation of cells, the extent of which varies between donors. However, late passage cultures in AS consistently display normal DNA copy numbers. Combined bisulfite restriction analysis and bisulfite sequencing show that although DNA methylation states are overall stable in culture, AS exhibits stronger propensity than FBS to maintain unmethylated states. Comparison of DNA methylation in BMMSCs with freshly isolated and cultured adipose stem cells (ASCs) also reveals that most genes unmethylated in both BMMSCs and ASCs in early passage are also unmethylated in uncultured ASCs. We conclude that (i) BMMSCs expanded in AS or FBS may display localized genetic alterations, (ii) AS tends to generate more consistent genomic backgrounds and DNA methylation patterns, and (iii) the unmethylated state of uncultured MSCs is more likely to be maintained in culture than the methylated state.     

LPS and cytokine-induced endothelial cell IL-6 release and ELAM-1 expression; involvement of serum.

In this in vitro study, the influence of serum-concentration, heat inactivation of the serum and the origin of the serum on the responsiveness of cultured human umbilical vein endothelial cells (HUVEC) to immunological challenges was investigated. Addition of human serum during stimulation with 1 microgram/ml bacterial lipopolysaccharide (LPS) increased endothelial cell ELAM-1 expression and interleukin (IL)-6 release five to ten-fold. Full endothelial cell responsiveness to LPS required 10 to 50% human serum and was largely abrogated after heating the serum for 30 minutes at 56 degrees C. Addition of newborn or fetal bovine serum instead of human serum, induced even higher IL-6 release and ELAM-1 expression in response to LPS, whilst heat-inactivation of these serum-batches only moderately decreased endothelial cell responses. Endothelial cell IL-6 release and ELAM-1 expression after stimulation with IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) were less influenced by heat inactivation of the serum and by omission of serum, whilst responses to PMA remained completely unaffected by such modifications in assay media. Finally, we demonstrated that endothelial cell IL-8 release also and ICAM-1 expression in response to LPS and cytokines were increased by addition of human serum, indicating that the use of serum-free assay media, or the use of media enriched with heat-inactivated (HI) human serum interferes with physiological endothelial cell responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mammalian tissue culture growth, viral replication, and cultivation using serum replacement factor

Variability, cost, and availability of fetal bovine serum are in question; thus, we examined whether using a serum replacement factor in multiple mammalian tissue cultures could support not only cell growth, but also viral replication, expression, and retention of phenotypic markers. By using a serum replacement in defined media, we demonstrated multipassage cell growth of several different cell lines and viral cultivation and replication equivalent to fetal bovine serum. Moreover, by supplementing media with a serum replacement factor we observed at least a 28% financial savings.     

Serologic analysis of human tumor antigens

Summary The immune adherence (IA) assay was used to measure serum reactivity of patients with melanoma and osteosarcoma against paired cell lines (tumor cells and normal skin fibroblasts obtained from the same individuals) grown in tissue culture. Sera from 224 patients with various stages of melanoma were compared with sera from 100 normal age- and sex-matched donors. None of the 18 stage I sera (0%), 23 of 166 (14%) stage II sera, 3 of 40 (7%) stage III sera, and 3 of 100 (3%) normal sera were highly reactive to a standard allogeneic melanoma-fibroblast pair. None of the sera exhibited unique activity against melanoma. There was no correlation between stage of melanoma and high serum reactivity, nor was this reactivity predictive of recurrence. Sera from 39 tumor-bearing osteosarcoma patients prior to amputation were compared with sera from 50 normal age-and sex-matched donors. Eight of 39 (21%) patient sera and 1 of 50 (2%) normal sera were highly reactive to an osteosarcoma-fibroblast pair. No sera had reactivity uniquely directed against osteosarcoma. Eight osteosarcoma and two melanoma patients were tested simultaneously against their autologous cultured tumor and skin cells. Only one of these patients exhibited high reactivity towards autologous cells, and this reactivity was equal against both osteosarcoma and normal cells. None of seven highly reactive osteosarcoma or six highly reactive melanoma sera had residual tumor-specific reactivity against allogeneic osteosarcoma or melanoma after absorption with cultured fibroblasts, cultured fetal fibroblasts, or fetal calf serum.