Bone formation using human adipose tissue-derived stromal cells and a biodegradable scaffold

Human adipose tissue, obtained by liposuction, was processed to obtain a fibroblast-like population of cells or adipose tissue-derived stromal cells (ATSCs). The ATSCs, as well as bone marrow-derived mesenchymal stem cells (BMSCs), have the capacity for renewal and the potential to differentiate into multiple lineages of mesenchymal tissues. These cells are capable of forming bone when implanted ectopically in an appropriate scaffold. The aim of this study was to evaluate a beta-tricalcium phosphate (beta-TCP) as a scaffold and to compare the potential of osteogenic differentiation of ATSCs with BMSCs. Both cell types were loaded into beta-TCP disk and cultured in an osteogenic induction medium. Optimal osteogenic differentiation in ATSCs in vitro, as determined by secretion of osteocalcin, scanning electron microscope, and histology, were obtained in the culturing with the beta-TCP disk. Furthermore, bone formation in vivo was examined by using the ATSC- or BMSC-loaded scaffolds in nude mice. The present results show that ATSCs have a similar ability to differentiate into osteoblasts and to synthesize bone in beta-TCP disk as have BMSCs.     

Osteogenic potential of human adipose tissue-derived stromal cells as an alternative stem cell source

Adult bone marrow contains mesenchymal stem cells (bone marrow-derived mesenchymal stem cells; BMSCs) which contribute to the generation of mesenchymal tissue such as bone, cartilage, muscle and adipose. However, using bone marrow as a source of stem cells has the limitation of a low cell number. An alternate source of adult stem cells that could be obtained in large quantities, under local anesthesia, with minimal discomfort would be advantageous. Human adipose tissue obtained by liposuction was processed to obtain a fibroblast-like population of cells or adipose tissue-derived stromal cells (ATSCs). In this study, we compared the osteogenic differentiation of ATSCs with that of BMSCs. Both cell types were cultured in atelocollagen honeycomb-shaped scaffolds with a membrane seal (ACHMS scaffold) for three-dimensional culturing in a specific osteogenic induction medium. Optimal osteogenic differentiation in both cell types, as determined by alkaline phosphatase cytochemistry, secretion of osteocalcin, mineral (calcium phosphate) deposition and scanning electron microscopy, was obtained with the same three-dimensional culture. Furthermore, osteoblastic lining in vivo was examined using ATSC-seeded or BMSC-seeded scaffolds in nude mice. The present results show that ATSCs have a similar ability to differentiate into osteoblasts to that of BMSCs.     

Role of apoptosis inhibition in various chondrocyte culture systems

Apoptosis may limit the utility of cell-based therapies for articular cartilage disorders. We tested the hypothesis that chondrocyte apoptosis can be reduced by optimizing the conditions employed to expand chondrocyte numbers in culture. Chondrocyte apoptosis was examined in monolayer and suspension culture, in the presence of fetal bovine serum (FBS) or autologous serum, and for culture periods of 2 or 4 days. The effect of these variables was assessed by measuring cell viability, Annexin V labeling and mitochondrial membrane potential. After 2 days of culture, the greatest increase in viable cell number (3.7-fold) occurred in monolayer cultures with autologous serum. After 4 days of culture, the greatest increase in cell number (9.0-fold) occurred in monolayer cultures supplemented with FBS. By Annexin V staining, the proportion of cells undergoing apoptosis after 2 days was not affected by the type of serum used or by culture in monolayer versus suspension. After 4 days of culture, the proportion of apoptotic cells was significantly reduced (35% to 13%, p<0.02) in suspension cultures with autologous serum. Apoptosis assessed by loss of mitochondrial membrane potential was decreased in the presence of autologous serum. These data suggest that suspension culture with autologous serum is useful in simultaneously maintaining cell proliferation and minimizing apoptosis in cultured human articular chondrocytes.     

Antigen-dependent immunotherapy of non-obese diabetic mice with immature dendritic cells

Immunotherapy can be used to induce immunological tolerance by a number of different protocols. During the last decade the ability to use tolerogenic dendritic cells (DCs) to prevent autoimmunity has received much attention. Many studies have attempted to use immature or semi‐mature DCs to induce tolerance in the non‐obese diabetic (NOD) mouse model of human type 1 diabetes. However, most studies to date have used protocols in which generation of DCs involved a culture step in fetal bovine serum (FBS)‐supplemented medium which may affect tolerance induction in a non‐specific fashion. Indeed, several studies have shown that DCs cultured in the presence of FBS will induce a powerful T helper type 2 (Th2) immune response towards FBS‐related antigens which can suppress an ongoing immune response. Hence, this may interfere with diabetes development in the NOD mouse by induction of immune deviation rather than by antigen‐specific tolerance. In order to test whether antigen‐specific tolerance induction by DC therapy is feasible in the NOD mouse, we therefore generated immature DCs using autologous serum [normal mouse serum (NMS)‐supplemented cultures] instead of FBS, and we show that these DCs can protect NOD mice from diabetes, if pulsed with insulin‐peptide antigens before adoptive transfer. Our data therefore support that DC therapy is able to prevent diabetes in the NOD mouse in an antigen‐specific manner.     

Comparison of a serum replacement (Omni Serum) and fetal bovine serum in cell cultures used to isolate herpes simplex virus from clinical specimens.

Traditionally, fetal bovine serum (FBS) has been the principal component in media used in the growth and maintenance of cell cultures. Recent shortages have affected the cost and availability of FBS to clinical laboratories. Furthermore, lot-to-lot variability can affect cell culture performance and growth. We evaluated a commercially available serum replacement (Omni Serum; Advanced Biotechnologies Inc., Columbia, Md.) for use in the growth of cell cultures and for use in maintenance media used for the isolation of herpes simplex virus from clinical specimens. Cells (rhabdomyosarcoma and mink lung) raised on 5% Omni Serum grew as well as those grown on 10% FBS. The sensitivity of the Omni-raised cells to herpes simplex virus that had been isolated from 111 clinical specimens was equal to that of the cells raised and maintained with FBS. Cells grown with 10% FBS and maintained with 2% Omni Serum displayed the same sensitivity and integrity in tubes (rhabdomyosarcoma and mink lung) and vials (MRC-5 cells) as cells grown with 10% FBS and maintained with 5% FBS. This study indicates that Omni Serum is an acceptable substitute for FBS in maintenance media for cell culture tubes and vials used for viral isolation from clinical specimens.     

Expression of telomerase extends the lifespan and enhances osteogenic differentiation of adipose tissue-derived stromal cells

Expression of TERT, the catalytic protein subunit of the telomerase complex, can be used to generate cell lines that expand indefinitely and retain multilineage potential. We have created immortal adipose stromal cell lines (ATSCs) by stably transducing nonhuman primate-derived ATSCs with a retroviral vector expressing TERT. Transduced cells (ATSC-TERT) had an increased level of telomerase activity and increased mean telomere length in the absence of malignant cellular transformation. Long-term culture of the ATSC-TERT cells demonstrated that the cells retain the ability to undergo differentiation along multiple lineages such as adipogenic, chondrogenic, and neurogenic. Untransduced cells demonstrated markedly reduced multilineage and self-renewal potentials after 12 passages in vitro. To determine the functional role of telomerase during osteogenesis, we examined osteogenic differentiation potential of ATSC-TERT cells in vitro. Compared with naive ATSCs, which typically begin to accumulate calcium after 3-4 weeks of induction by osteogenic differentiation medium, ATSC-TERT cells were found to accumulate significant amounts of calcium after only 1 week of culture in osteogenic induction medium. The cells have increased production of osteoblastic markers, such as AP2, osteoblast-specific factor 2, chondroitin sulfate proteoglycan 4, and the tumor necrosis factor receptor superfamily, compared with control ATSCs, indicating that telomerase expression may aid in maintaining the osteogenic stem cell pool during in vitro expansion. These results show that ectopic expression of the telomerase gene in nonhuman primate ATSCs prevents senescence-associated impairment of osteoblast functions and that telomerase therapy may be a useful strategy for bone regeneration and repair.     

Evaluation of porcine aortic valve interstitial cell activity using different serum types in two- and three-dimensional culture

Unlike established cell lines used in the biotechnology industry, primary cells used in tissue engineering require culture media to be supplemented with serum. The most common serum is fetal bovine serum (FBS); however, FBS is expensive, negatively affecting process economics. Less-costly alternative sera are commercially available, but their efficacy has not been documented. Therefore, bovine calf serum (BCS), bovine growth serum (BGS), and newborn calf serum (NCS) were compared with FBS. Porcine aortic valve interstitial cells (VICs) were cultured as 2-dimensional (2-D) monolayers or as 3-dimensional (3-D) collagen gels using medium supplemented with 10% FBS, BGS, BCS, or NCS. No significant difference was seen in cellular activity between VICs cultured in BCS and those cultured in FBS in 2-D cultures, whereas cells cultured in BGS and NCS had significantly lower specific growth rates coupled with markedly higher metabolic activity than cells cultured in FBS. No statistically significant differences were seen in cellular activity between any of the sera when cells were cultured in 3-D constructs. In conclusion, BCS is a suitable alternative to FBS for the 2-D and 3-D culture of VICs, which may be used to develop a tissue-engineered valve.     

Tissue engineering of articular cartilage with autologous cultured adipose tissue-derived stromal cells using atelocollagen honeycomb-shaped scaffold with a membrane sealing in rabbits

Adipose tissue derived stromal cells (ATSCs), which were isolated from adipose tissue of rabbit, have shown to possess multipotential, that is, they differentiate into osteoblasts and adipocytes in plate-culturing and into chondrocytes in an established aggregate culture using defined differentiation-inductive medium. The aim of this study was to evaluate the utility of ATSCs in tissue engineering procedures for repair of articular cartilage-defects using the atelocollagen honeycomb-shaped scaffold with a membrane sealing (ACHMS-scaffold). We intended to repair full-thickness articular cartilage defects in rabbit knees using autologously cultured ATSCs embedded in the ACHMS-scaffold. ATSCs were incubated within the ACHMS-scaffold to allow a high density and three-dimensional culture with control medium. An articular cartilage defect was created on the patellar groove of the femur, and the defect was filled with the ATSCs-containing ACHMS-scaffold, ACHMS-scaffold alone, or empty (control). Twelve weeks after the operation, the histological analyses showed that only the defects treated with the ATSCs-containing ACHMS-scaffold were filled with reparative hyaline cartilage, highly expressed Type II collagen. These results indicate that transplantation of autologous ATSCs-containing ACHMS-scaffold is effective in repairing articular cartilage defects.     

Differentiation of myoblasts in serum-free media: effects of modified media are cell line-specific

Myoblast cell lines are grown and differentiated readily in cell culture. Two cell lines typically used for investigating the growth and differentiation of muscle are the mouse cell line C2C12 and the rat cell line L6. The differentiation of these cells in vitro requires a switch from a serum-rich medium to a less rich medium after the cells have reached confluence. Since the components present in serum are not well characterized, the use of a better defined medium for these studies was investigated. C2C12 and L6 myoblasts were differentiated in both serum-containing and serum-free media. The differentiation state of these cultures was then tested both microscopically and biochemically. Cultures were checked for myotube formation, the activity of creatine phosphokinase and the presence of sarcomeric actin. In C2C12 cells, the extent of differentiation was greater in the serum-free than in the serum-containing system. In both media types, the C2C12 cells produced sarcomeric actin, showing the presence of sarcomere structure in the myotubes. In L6 cells, however, myotubes were readily formed in medium containing 2% horse serum, but not in the serum-free system. In addition, the ability of C2C12 cells to differentiate on substrates coated with extracellular matrix proteins was shown to be media-dependent. The presence of extracellular matrix proteins did not enable L6 cells to form myotubes when cultured in serum-free media. Primary cultures of chick myoblasts were able to differentiate in both media tested, with Dulbecco's modified Eagle medium containing horse serum being a more efficient medium for cell fusion. This study shows a divergence in muscle cell line responses in three cell lines, two of which are typically used as 'model systems' for understanding muscle growth and development.     

新生小鼠端脑神经干细胞可诱导分化为运动神经元

目的 探讨新生小鼠端脑神经干细胞诱导分化为运动神经元的可能性,并探索新的运动神经元诱导因子.方法 用悬浮培养法从新生小鼠端脑分离培养神经干细胞,按诱导因素的不同分为3组:组1为对照组,诱导因素为生长培养基+5%胎牛血清(FBS);组2为诱导因子组,诱导因素为生长培养基+5% FBS+视黄酸(RA)+Shh+联丁酰基环磷酸腺苷(dbcAMP);组3为骨骼肌细胞培养液组,诱导因素为骨骼肌细胞生长过的培养液.用双重免疫荧光方法检测分化细胞的微管相关蛋白2(MAP2)和同源蛋白(HB9)的表达,以验证运动神经元的分化,每组随机选取12个标本计数.结果 分化培养中检测到MAP2和HB9共表达的运动神经元,组1的运动神经元分化比例为1%;组2的分化比例为4.7%;组3的分化比例为2.9%.与组1相比有显著的统计学差异.结论 新生小鼠端脑神经干细胞能诱导分化为运动神经元;骨骼肌细胞可能分泌运动神经元诱导因子.     

Fetal bovine serum contains an inhibitor of interleukin-1

The keratinocyte cell line COLO-16 constitutively produces factors with interleukin-1 (IL-1) activity including IL-1 alpha and IL-1 beta. IL-1 activity assayed by thymocyte proliferation from cell supernatants was 20-50% less if cells were maintained in media containing 10% fetal bovine serum (FBS) compared to media without serum 24 h prior to harvest. The increased IL-1 activity in supernatants from cells in serum free media was not due to increased cellular levels of IL-1 alpha or IL-1 beta mRNA. Similarly, IL-1 activity recovered from conditioned supernatants of COS cells transfected with expression vectors containing IL-1 beta cDNA was approximately 22-45% less in cells grown in 20% FBS medium compared to similar cultures grown for 3 days post transfection in 1% FBS. When serial dilutions of recombinant IL-1 were made in buffer containing 10% FBS and assayed by a thymocyte proliferation method, a 30-50% decrease in activity was observed. IL-1 activity was also measured by its ability to induce prostaglandin E2 synthesis by fibroblasts. When COS conditioned supernatants were applied to fibroblast cultures there was 30% less prostaglandin E2 activity from fibroblasts treated with COS supernatants containing 20% FBS, compared to supernatants containing 1% FBS. The inhibitor molecule was partially purified by gel filtration and found to have a molecular weight of approximately 85,000. The presence of FBS in cell-conditioned media significantly reduces the sensitivity of IL-1 detection by bioassay techniques.     

Umbilical cord tissue-derived mesenchymal stem cells grow best under GMP-compliant culture conditions and maintain their phenotypic and functional properties

Mesenchymal stem cells (MSCs) are fibroblast-like multipotent stem cells that can differentiate into cell types of mesenchymal origin. Because of their immune properties and differentiation, potential MSCs are discussed for the use in tissue regeneration and tolerance induction in transplant medicine. This cell type can easily be obtained from the umbilical cord tissue (UCMSC) without medical intervention. Standard culture conditions include fetal bovine serum (FBS) which may not be approved for clinical settings. Here, we analyzed the phenotypic and functional properties of UCMSC under xeno-free (XF, containing GMP-certified human serum) and serum-free (SF) culture conditions in comparison with standard UCMSC cultures. Phenotypically, UCMSC showed no differences in the expression of mesenchymal markers or differentiation capacity. Functionally, XF and SF-cultured UCMSC have comparable adipogenic, osteogenic, and endothelial differentiation potential. Interestingly, the UCMSC-mediated suppression of T cell proliferation in an allogeneic mixed lymphocyte reaction (MLR) is more effective in XF and SF media than in standard FBS-containing cultures. Regarding the mechanism of action of MLR suppression, transwell experiments revealed that in neither UCMSC culture a direct cell-cell contact is necessary for inhibiting T cell proliferation, and that the major effector molecule is prostaglandin E₂ (PGE₂). Taken together, GMP-compliant growth media qualify for long-term cultures of UCMSC which is important for a future clinical study design in regenerative and transplant medicine.     

The morphological and karyological characteristics of MDCK and vero (B) cells cultures on plant hydrolyzate-based nutrient media

MDCK and Vero (B) cell cultures were propagated during 10 passages in the experimental nutrient media containing the soybean powder hydrolyzate prepared using trypsin and bromelain enzymes and the rice powder hydrolysate prepared with trypsin and in the control DMEM and SFM4 MegaVir media. The karyological, morphological, and proliferative characteristics of continuous cultures were examined and compared. The experimental media supplied with 3% fetal bovine serum (FBS) (Gibco, U.S.A.) showed high growth-enhancing properties and failed to affect their morphology. After propagated during 10 passages in the experimental media preserved a stable karyotype. MDCK cell cultures in the nutrient media based on rice and soybean powder hydrolyzates low (2%) in FBS caused no substantial changes in the proliferation indices and morphological and karyological characteristics of cell cultures.     

Use of serum substitutes for the growth of four cell lines commonly used for virus isolation

Four serum extenders (Opti-MEM I, BioRich, Excyte I and Excyte III) and two serum substitutes (Omni Serum and Serum Plus) were evaluated for the reduction or replacement of FBS for the growth and maintenance of four representative cell lines used for virus isolation. MRC-5, human fetal foreskin fibroblast (HF), A549 and Hep-2 cells were seeded into culture flasks containing MEM with 10% FBS or with the serum extenders or substitutes and subcultured into 16 x 125 mm glass tubes and 1-dram shell vials. Only cells cultured with Opti-MEM I and Omni Serum grew consistently in tubes and vials and these reagents were compared to FBS for viral isolation and detection. Laboratory stocks of CMV, HSV, VZV, adenovirus (Ad) and RSV were tested. In HF and MRC-5 cells, CMV was isolated in cells cultured in either Opti-MEM I or Omni Serum before CPE appeared in cultures containing FBS. Ad and VZV CPE were observed first in HF and MRC-5 cells containing Omni Serum. HSV CPE was seen at the same time in HF and MRC-5 cells with all 3 media. HSV and Ad CPE in A549 cells were also seen at the same time in all 3 media. RSV and Ad CPE in Hep-2 cells were observed first in FBS containing media. Both Opti-MEM I and Omni Serum performed well as substitutes for FBS for growth of stock viruses without loss of cell sensitivity.     

Secretion of insulin-like growth factor (IGF)-I and -II and IGF binding proteins (IGFBPs) in fetal stromal-vascular (S-V) cell cultures obtained before and after the onset of adipogenesis in vivo

The present study examined the influence of dexamethasone (DEX) treatment on preadipocyte differentiation and insulin-like growth factor binding protein (IGFBP) secretion in stromal-vascular (S-V) cell cultures established from subcutaneous adipose tissue obtained from nine 75 day and four 50 day pig fetuses. Cultures of S-V cells from four young pigs (5-7 days old) were also studied. Each fetal S-V cell culture represented 1 pool of S-V cells/dam. Cultures were seeded and plated in 10% FBS from day 0-3 and treated with insulin (ITS) + 10 nM DEX from day 3-6 (late DEX treatment). Alternatively, cultures were seeded and plated in 10% FBS + 80 nM DEX from day 0-3 and treated with insulin alone from day 3-6 (early DEX treatment). Conditioned media was collected on day 6 of culture after 3 days of conditioning, and prepared for subsequent 125I-IGF-I ligand blot analysis for IGFBPs and RIA for IGF-I and IGF-II. Early and late DEX increased (P<0.05) preadipocyte (AD-3+) recruitment but only early DEX increased preadipocyte differentiation (lipid + and C/EBP alpha+) by day 6 in S-V cultures from 75 day fetuses. Levels of IGFBP-2, IGFBP-4, IGF-I and IGF-II in media conditioned by 75 day fetal S-V cultures were not influenced by late DEX. However, late DEX reduced levels of 29 kDa IGFBPs and markedly increased (P<0.05) IGFBP-3 levels in 75 day S-V media. Late DEX also markedly increased (P<0.05) IGFBP-3 levels in 50 day S-V media but had little influence on other IGFBPs. Early DEX treatment increased (P<0.05) IGFBP-4 levels in 75 day S-V media but had little to no influence on levels of IGF-I, IGF-II and other IGFBPs. These studies indicate that IGFBP-4 may regulate local metabolism during preadipocyte differentiation, whereas IGFBP-3 may antagonize preadipocyte differentiation by targeting IGF-I away from differentiating cells and towards growing cells.     

Identification and purification of mesodermal progenitor cells from human adult bone marrow

Bone marrow-derived mesodermal stem cells may differentiate toward several lines and are easily cultured in vitro. Some putative progenitors of these cells have been described in both humans and mice. Here, we describe a new mesodermal progenitor population [mesodermal progenitors cells (MPCs)] able to differentiate into mesenchymal cells upon appropriate culture conditions. When cultured in presence of autologous serum, these cells are strongly adherent to plastic, resistant to trypsin detachment, and resting. Mesodermal progenitor cells may be pulsed to proliferate and differentiate by substituting autologous serum for human cord blood serum or fetal calf serum. By these methods cells proliferate and differentiate toward mesenchymal cells and thus may further differentiate into osteoblats, chondrocytes, or adipocytes. Moreover MPCs are capable to differentiate in endothelial cells (ECs) showing characteristics similar to microvessel endothelium cells. Mesodermal progenitors cells have a defined phenotype and carry embryonic markers not present in mesenchymal cells. Moreover MPCs strongly express aldehyde dehydrogenase activity, usually present in hematopoietic precursors but absent in mesenchymal cells. When these progenitors are pulsed to differentiate, they lose these markers and acquire the mesenchymal ones. Interestingly, mesenchymal cells may not be induced to back differentiate into MPCs. Our results demonstrate the adult serum role in maintaining pluripotent mesodermal precursors and allow isolation of these cells. After purification, MPCs may be pulsed to proliferate in a very large scale and then induced to differentiate, thus possibly allowing their use in regenerative medicine.     

比较牛、兔血清对小鼠B淋巴细胞杂交瘤的影响

在制备抗SRBC McAb时,对比试用进口胎牛血清、自制兔血清和国产新生牛血清,并就不同种属血清对小鼠骨髓癌细胞的生长和融合率进行比较,提示胎牛血清和自制兔血清优于新生牛血清。     

Generation of natural killer cells and lymphokine-activated killer cells in human AB serum or fetal bovine serum

Interleukin 2 (IL-2) can induce or enhance the cytotoxic activity of natural killer (NK) cells and lymphokine-activated killer (LAK) cells. The effects of fetal bovine serum (FBS) and human AB serum (HABS) on the IL-2-induced NK cell and LAK cell activities of large granular lymphocytes (LGL) were measured, respectively, against the NK-sensitive cell line K562 and the LAK-sensitive cell line Daudi. FBS and HABS were essentially equivalent in their effects on IL-2-dependent NK activity with prolonged culture. However, with prolonged incubation from 1 to 5 days of PBMC in the presence of IL-2, there was considerably less generation of LAK cell activity in FBS (Day 3: 5.3 +/- 3.4 LU/10(7) cells) compared to HABS (Day 3: 44.6 +/- 4.2 LU/10(7) cells) (P less than 0.05). These differences in IL-2-dependent LAK cell generation did not appear to be due to the lot of the FBS or to activating factors present in individual samples of HABS. Similarly, the suppressive effects of FBS could not be reversed with increasing concentrations of IL-2 ranging from 10 to 100 U/ml. Importantly, the presence of FBS in the cultures resulted in more cell death (15.9 +/- 5.6%) at 4 days of culture compared to HABS (1.8 +/- 1.0%) (P less than 0.05). These results suggest that FBS may inhibit generation of LAK effector cells, but not NK cells in cultures containing IL-2 and that the use of HABS as a culture supplement is preferable to FBS in studies of human LAK cell function.