A Synthetic,Xeno-Free Peptide Surface for Expansion and Directed Differentiation of Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells have the potential to become an unlimited cell source for cell replacement therapy. The realization of this potential, however, depends on the availability of culture methods that are robust, scalable, and use chemically defined materials. Despite significant advances in hiPSC technologies, the expansion of hiPSCs relies upon the use of animal-derived extracellular matrix extracts, such as Matrigel, which raises safety concerns over the use of these products. In this work, we investigated the feasibility of expanding and differentiating hiPSCs on a chemically defined, xeno-free synthetic peptide substrate, i.e. Corning Synthemax® Surface. We demonstrated that the Synthemax Surface supports the attachment, spreading, and proliferation of hiPSCs, as well as hiPSCs’ lineage-specific differentiation. hiPSCs colonies grown on Synthemax Surfaces exhibit less spread and more compact morphology compared to cells grown on Matrigel™. The cytoskeleton characterization of hiPSCs grown on the Synthemax Surface revealed formation of denser actin filaments in the cell-cell interface. The down-regulation of vinculin and up-regulation of zyxin expression were also observed in hiPSCs grown on the Synthemax Surface. Further examination of cell-ECM interaction revealed that hiPSCs grown on the Synthemax Surface primarily utilize α_vβ₅ integrins to mediate attachment to the substrate, whereas multiple integrins are involved in cell attachment to Matrigel. Finally, hiPSCs can be maintained undifferentiated on the Synthemax Surface for more than ten passages. These studies provide a novel approach for expansion of hiPSCs using synthetic peptide engineered surface as a substrate to avoid a potential risk of contamination and lot-to-lot variability with animal derived materials.     

Stem cell-derived extracellular matrix enables survival and multilineage differentiation within superporous hydrogels

Hydrophilic poly(ethylene glycol) diacrylate (PEGDA) hydrogel surfaces resist protein adsorption and are generally thought to be unsuitable for anchorage-dependent cells to adhere. Intriguingly, our previous findings revealed that PEGDA superporous hydrogel scaffolds (SPHs) allow anchorage of bone marrow derived human mesenchymal stem cells (hMSCs) and support their long-term survival. Therefore, we hypothesized that the physicochemical characteristics of the scaffold impart properties that could foster cellular responses. We examined if hMSCs alter their microenvironment to allow cell attachment by synthesizing their own extracellular matrix (ECM) proteins. Immunofluorescence staining revealed extensive expression of collagen type I, collagen type IV, laminin, and fibronectin within hMSC-seeded SPHs by the end of the third week. Whether cultured in serum-free or serum-supplemented medium, hMSC ECM protein gene expression patterns exhibited no substantial changes. The presence of serum proteins is required for initial anchorage of hMSCs within the SPHs but not for the hMSC survival after 24 h. In contrast to 2D expansion on tissue culture plastic (TCP), hMSCs cultured within SPHs proliferate similarly in the presence or absence of serum. To test whether hMSCs retain their undifferentiated state within the SPHs, cell-seeded constructs were cultured for 3 weeks in stem cell maintenance medium and the expression of hMSC-specific cell surface markers were evaluated by flow cytometry. CD105, CD90, CD73, and CD44 were present to a similar extent in the SPH and in 2D monolayer culture. We further demonstrated multilineage potential of hMSCs grown in the PEGDA SPHs, whereby differentiation into osteoblasts, chondrocytes, and adipocytes could be induced. The present study demonstrates the potential of hMSCs to alter the "blank" PEGDA environment to a milieu conducive to cell growth and multilineage differentiation by secreting adhesive ECM proteins within the porous network of the SPH scaffolds.     

Serum-converted platelet lysate can substitute for fetal bovine serum in human mesenchymal stromal cell cultures

Background aimsFetal bovine serum (FBS) is commonly used as a serum supplement for culturing human mesenchymal stromal cells (hMSCs). However, human cells grown in FBS, especially for extended periods, risk potential exposure to bovine immunogenic proteins and infectious agents. To address this issue, we investigated the ability of a novel human platelet serum supplement to substitute for FBS in hMSC cultures.MethodsPlatelet lysate-serum (PL-serum) was converted from platelet lysate-plasma (PL-plasma) that was manufactured from pooled platelet-rich plasma (PRP) apheresis units. Growth factor levels and the number of residual intact platelets in PL-serum and PL-plasma were compared with enzyme-linked immunosorbent assays and flow cytometry, respectively. Proliferation responses of hMSCs cultured in PL-serum, PL-plasma, or FBS were assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the immunophenotype of harvested hMSCs was evaluated by flow cytometry and tri-lineage differentiation potential was evaluated by assessing adipogenic, osteogenic and chondrogenic development.ResultsSelected growth factor levels in PL-serum were not significantly different from PL-plasma (P > 0.05). hMSC cultures supplemented with PL-serum had comparable growth kinetics to PL-plasma, and hMSC yields were consistently greater than with FBS. hMSCs harvested from cultures supplemented with PL-serum, PL-plasma or FBS had similar cell surface phenotypes and maintained tri-lineage differentiation potential.ConclusionsPL-serum, similar to PL-plasma, can substitute for FBS in hMSC cultures. Use of PL-serum, in contrast to PL-plasma, has an added advantage of not requiring addition of a xenogeneic source of heparin, providing a completely xeno-free culture medium.     

Human mesenchymal stem cells from the umbilical cord matrix: Successful isolation and ex vivo expansion using serum-/xeno-free culture media

Mesenchymal stem cells (MSC) could potentially be applied in therapeutic settings due to their multilineage differentiation ability, immunomodulatory properties, as well as their trophic activity. The umbilical cord matrix (UCM) represents a promising source of MSC for biomedical applications. The number of cells isloated per umbilical cord (UC) unit is limited and ex vivo expansion is imperative in order to reach clinically meaningful cell numbers. The limitations of poorly defined reagents (e.g. fetal bovine serum, which is commonly used as a supplement for human MSC expansion) make the use of serum-/xeno-free conditions mandatory. We demonstrated the feasibility of isolating UCM-MSC by plastic adherence using serum-/xeno-free culture medium following enzymatic digestion of UCs, with a 100% success rate. 2.6 ± 0.21 × 10⁵ cells were isolated per UC unit, of which 1.9 ± 0.21 × 10⁵ were MSC-like cells expressing CD73, CD90, and CD105. When compared to adult sources (bone marrow-derived MSC and adipose-derived stem/stromal cells), UCM-MSC displayed a similar immunophenotype and similar multilineage differentiation ability, while demonstrating a higher expansion potential (average fold increase of 7.4 for serum-containing culture medium and 11.0 for xeno-free culture medium (P3-P6)). The isolation and expansion of UCM-MSC under defined serum-/xeno-free conditions contributes to safer and more effective MSC cellular products, boosting the usefulness of MSC in cellular therapy and tissue engineering.     

A Defined and Xeno-Free Culture Method Enabling the Establishment of Clinical-Grade Human Embryonic,Induced Pluripotent and Adipose Stem Cells

Background

The growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable.

Methodology/Principal Findings

Here, we report the development of a fully defined xeno-free medium (RegES), capable of supporting the expansion of human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC) and adipose stem cells (ASC). We describe the use of the xeno-free medium in the derivation and long-term (>80 passages) culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS), while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed.

Conclusion/Significance

Our results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation described herein has the potential to be further optimized for specific applications relating to establishment, expansion and differentiation of various stem cell types.     

MSCs Conditioned Media and Umbilical Cord Blood Plasma Metabolomics and Composition

Human mesenchymal stem cells (hMSCs) from umbilical cord (UC) blood (UCB) and matrix are tested clinically for a variety of pathologies but in vitro expansion using culture media containing fetal bovine serum (FBS) is essential to achieve appropriate cell numbers for clinical use. Human UCB plasma (hUCBP) can be used as a supplement for hMSCs culture, since UCB is rich in soluble growth factors and due to worldwide increased number of cryopreserved UCB units in public and private banks, without the disadvantages listed for FBS. On the other hand, the culture media enriched in growth factors produced by these hMSCs in expansion (Conditioned medium - CM) can be an alternative to hMSCs application. The CM of the hMSCs from the UC might be a better therapeutic option compared to cell transplantation, as it can benefit from the local tissue response to the secreted molecules without the difficulties and complications associated to the engraftment of the allo- or xeno-transplanted cells. These facts drove us to know the detailed composition of the hUCBP and CM, by ¹H-NMR and Multiplexing LASER Bead Technology. hUCBP is an adequate alternative for the FBS and the CM and hUCBP are important sources of growth factors, which can be used in MSCs-based therapies. Some of the major proliferative, chemotactic and immunomodulatory soluble factors (TGF-β, G-CSF, GM-CSF, MCP-1, IL-6, IL-8) were detected in high concentrations in CM and even higher in hUCBP. The results from ¹H-NMR spectroscopic analysis of CM endorsed a better understanding of hMSCs metabolism during in vitro culture, and the relative composition of several metabolites present in CM and hUCBP was obtained. The data reinforces the potential use of hUCBP and CM in tissue regeneration and focus the possible use of hUCBP as a substitute for the FBS used in hMSCs in vitro culture.     

Growth and functional harvesting of human mesenchymal stromal cells cultured on a microcarrier‐based system

Human mesenchymal stromal cells (hMSCs) cells are attractive for applications in tissue engineering and cell therapy. Because of the low availability of hMSCs in tissues and the high doses of hMSCs necessary for infusion, scalable and cost‐effective technologies for in vitro cell expansion are needed to produce MSCs while maintaining their functional, immunophenotypic and cytogenetic characteristics. Microcarrier‐based culture systems are a good alternative to traditional systems for hMSC expansion. The aim of the present study was to develop a scalable bioprocess for the expansion of human bone marrow mesenchymal stromal cells (hBM‐MSCs) on microcarriers to optimize growth and functional harvesting. In general, the results obtained demonstrated the feasibility of expanding hBM‐MSCs using microcarrier technology. The maximum cell concentration (n = 5) was ～4.82 ± 1.18 × 10⁵ cell mL^?1 at day 7, representing a 3.9‐fold increase relative to the amount of inoculated cells. At the end of culture, 87.2% of the cells could be harvested (viability = 95%). Cell metabolism analysis revealed that there was no depletion of important nutrients such as glucose and glutamine during culture, and neither lactate nor ammonia byproducts were formed at inhibitory concentrations. The cells that were recovered after the expansion retained their immunophenotypic and functional characteristics. These results represent an important step toward the implementation of a GMP‐compliant large‐scale production system for hMSCs for cellular therapy. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:889–895, 2014     

Defined Essential 8? Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems

Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm² of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x10⁶ cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells.     

Isolation and Characterization of Human Dental Pulp Derived Stem Cells by Using Media Containing Low Human Serum Percentage as Clinical Grade Substitutes for Bovine Serum

Adult stem cells have been proposed as an alternative to embryonic stem cells to study multilineage differentiation in vitro and to use in therapy. Current culture media for isolation and expansion of adult stem cells require the use of large amounts of animal sera, but animal-derived culture reagents give rise to some questions due to the real possibility of infections and severe immune reactions. For these reasons a clinical grade substitute to animal sera is needed. We tested the isolation, proliferation, morphology, stemness related marker expression, and osteoblastic differentiation potential of Dental Pulp Stem Cells (DPSC) in a chemically defined medium containing a low percentage of human serum, 1.25%, in comparison to a medium containing 10% Fetal Bovine Serum (FBS). DPSCs cultured in presence of our isolation/proliferation medium added with low HS percentage were obtained without immune-selection methods and showed high uniformity in the expression of stem cell markers, proliferated at higher rate, and demonstrated comparable osteoblastic potential with respect to DPSCs cultured in 10% FBS. In this study we demonstrated that a chemically defined medium added with low HS percentage, derived from autologous and heterologous sources, could be a valid substitute to FBS-containing media and should be helpful for adult stem cells clinical application.     

Human platelet lysate stimulates high-passage and senescent human multipotent mesenchymal stromal cell growth and rejuvenation in vitro

Background aimsMultipotent mesenchymal stromal cells (MSCs) are clinically useful because of their immunomodulatory and regenerative properties, but MSC therapies are limited by the loss of self-renewal and cell plasticity associated with ex vivo expansion culture and, on transplantation, increased immunogenicity from xenogen exposure during culture. Recently, pooled human platelet lysate (hPL) has been used as a culture supplement to promote MSC growth; however, the effects of hPL on MSCs after fetal bovine serum (FBS) exposure remain unknown.MethodsMSCs were cultured in medium containing FBS or hPL for up to 16 passages, and cell size, doubling time and immunophenotype were determined. MSC senescence was assessed by means of a fluorometric assay for endogenous β-galactosidase expression. MSCs cultured with FBS for different numbers of passages were switched to hPL conditions to evaluate the ability of hPL to “rescue” the proliferative capacity of MSCs.ResultshPL culture resulted in more rapid cell proliferation at earlier passages (passage 5 or earlier) than remove FBS; by day 4, hPL (5%) yielded an MSC doubling time of 1.28 days compared with 1.52 days in 16% FBS. MSCs cultured first in FBS and switched to hPL proliferated more and demonstrated less β-galactosidase production and smaller cell sizes than remove MSCs continuously propagated in FBS.ConclusionshPL enables rapid expansion of MSCs without adversely affecting immunophenotype. hPL culture of aged and senescent MSCs demonstrated cellular rejuvenation, reflected by decreased doubling time and smaller cell size. These results suggest that expansion of MSCs in hPL after FBS exposure can enhance cell phenotype and proliferative capacity.     

Growth factor-defined culture medium for human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) are potential cellular sources of therapeutic stem cells as they have the ability to proliferate and differentiate into a wide array of mesenchymal cell types such as osteoblasts, chondroblasts and adipocytes. hMSCs have been used clinically to treat patients with graft vs. host disease, osteogenesis imperfect, or alveolar cleft, suggesting that transplantation of hMSCs is comparatively safe as a stem cell-based therapy. However, conventional culture medium for hMSCs contains fetal bovine serum (FBS). In the present study, we developed a growth factor-defined, serum-free medium for culturing hMSCs. Under these conditions, TGF-beta1 promoted proliferation of hMSCs. The expanded hMSC population expressed the human pluripotency markers SSEA-3, -4, NANOG, OCT3/4 and SOX2. Furthermore, double positive cells for SSEA-3 and a mesenchymal cell marker, CD105, were detected in the population. The potential to differentiate into osteoblasts and adipocytes was confirmed. This work provides a useful tool to understand the basic biological properties of hMSCs in culture.     

Human macrovascular endothelial cells: Optimization of culture conditions

Summary The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage.     

Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells.     

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.     

Differentiation of human embryonic stem cells to hepatocyte-like cells on a new developed xeno-free extracellular matrix

Human embryonic stem cells (hESCs) provide a new source for hepatocyte production in translational medicine and cell replacement therapy. The reported hESC-derived hepatocyte-like cells (HLCs) were commonly generated on Matrigel, a mouse cell line-derived extracellular matrix (ECM). Here, we performed the hepatic lineage differentiation of hESCs following a stepwise application of growth factors on a newly developed serum- and xeno-free, simple and cost-benefit ECM, designated “RoGel,” which generated from a modified conditioned medium of human fibroblasts. In comparison with Matrigel, the differentiated HLCs on both ECMs expressed similar levels of hepatocyte-specific genes, secreted α-fetoprotein, and metabolized ammonia, showed glycogen storage activity as well as low-density lipoprotein and indocyanine green uptake. The transplantation of hESC–HLCs into the carbon tetrachloride-injured liver demonstrated incorporation of the cells into the host mouse liver and the expression of albumin. The results suggest that the xeno-free and cost-benefit matrix may be applicable in bioartificial livers and also may facilitating a clinical application of human pluripotent stem cell-derived hepatocytes in the future.     

Xeno-Free Defined Conditions for Culture of Human Embryonic Stem Cells,Neural Stem Cells and Dopaminergic Neurons Derived from Them

Background

Human embryonic stem cells (hESCs) may provide an invaluable resource for regenerative medicine. To move hESCs towards the clinic it is important that cells with therapeutic potential be reproducibly generated under completely defined conditions.

Methodology/Principal Findings

Here we report a four-step scalable process that is readily transferable to a Good Manufacture Practice (GMP) facility for the production of functional dopaminergic neurons from hESCs for potential clinical uses. We show that each of the steps (propagation of ESC→generation of neural stem cells (NSC)→induction of dopaminergic precursors→maturation of dopaminergic neurons) could utilize xeno-free defined media and substrate, and that cells could be stored at intermediate stages in the process without losing their functional ability. Neurons generated by this process expressed midbrain and A9 dopaminergic markers and could be transplanted at an appropriate time point in development to survive after transplant.

Conclusions/Significance

hESCs and NSCs can be maintained in xeno-free defined media for a prolonged period of time while retaining their ability to differentiate into authentic dopaminergic neurons. Our defined medium system provides a path to a scalable GMP-applicable process of generation of dopaminergic neurons from hESCs for therapeutic applications, and a ready source of large numbers of neurons for potential screening applications.     

N‐isopropylacrylamide‐based thermoresponsive polyelectrolyte multilayer films for human mesenchymal stem cell expansion

Human mesenchymal stem cells (hMSCs) are colony‐forming unit fibroblasts (CFU‐F) derived from adult bone marrow and have significant potential for many cell‐based tissue‐engineering applications. Their therapeutic potential, however, is restricted by their diminishing plasticity as they are expanded in culture. In this study, we used N‐isopropylacrylamide (NIPAM)‐based thermoresponsive polyelectrolyte multilayer (N‐PEMU) films as culture substrates to support hMSC expansion and evaluated their effects on cell properties. The N‐PEMU films were made via layer‐by‐layer adsorption of thermoresponsive monomers copolymerized with charged monomers, positively charged allylamine hydrochloride (PAH), or negatively charged styrene sulfonic acid (PSS) and compared to fetal bovine serum (FBS) coated surfaces. Surface charges were shown to alter the extracellular matrix (ECM) structure and subsequently regulate hMSC responses including adhesion, proliferation, integrin expression, detachment, and colony forming ability. The positively charged thermal responsive surfaces improved cell adhesion and growth in a range comparable to control surfaces while maintaining significantly higher CFU‐F forming ability. Immunostaining and Western blot results indicate that the improved cell adhesion and growth on the positively charged surfaces resulted from the elevated adhesion of ECM proteins such as fibronectin on the positively charge surfaces. These results demonstrate that the layer‐by‐layer approach is an efficient way to form PNIPAM‐based thermal responsive surfaces for hMSC growth and removal without enzymatic treatment. The results also show that surface charge regulates ECM adhesion, which in turn influences not only cell adhesion but also CFU‐forming ability and their multi‐lineage differentiation potential. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Perfusion affects the tissue developmental patterns of human mesenchymal stem cells in 3D scaffolds

Human mesenchymal stem cells (hMSCs) developed in three‐dimensional (3D) scaffolds are significantly affected by culture conditions. We hypothesized that the hydrodynamic forces generated in perfusion bioreactors significantly affected hMSC functionality in 3D scaffolds by shaping the extracellular matrix (ECM) proteins. In this study, hMSCs were grown in 3D poly(ethylene terephthalate) (PET) scaffolds in static and a parallel perfusion system under similar initial conditions for up to 35 days. Results demonstrated that even at very low media velocities (O [10^?4 cm/sec]), perfusion cultures affected the ability of hMSCs to form an organized ECM network as illustrated by the immunostaining of collagen I and laminin fibrous structure. The change in the ECM microenvironment consequently influenced the nuclear shape. The hMSCs grown at the lower surface of static culture displayed a 15.2 times higher nuclear elongation than those at the upper surface, whereas cells grown in the perfusion bioreactor displayed uniform spherical nuclei on both surfaces. The difference in ECM organization and nuclear morphology associated with gene expression and differentiation characteristics of hMSCs. The cells exhibited lower CFU‐F colony forming ability and decreased expressions of stem‐cell genes of Rex‐1 and Oct‐4, implying a less primitive stem‐cell phenotype was maintained in the perfusion culture relative to the static culture conditions. The significantly higher expression level of osteonectin gene in the perfusion culture at day 28 indicated an upregulation of osteogenic ability of hMSCs. The study highlights the critical role of dynamic culture conditions on 3D hMSC construct development and properties. J. Cell. Physiol. 219: 421–429, 2009. © 2009 Wiley‐Liss, Inc.     

Mouse embryonic stem cell expansion in a microcarrier-based stirred culture system

Embryonic stem (ES) cells have the ability to differentiate in vitro into a wide variety of cell types with potential applications for tissue regeneration. However, a large number of cells are required, thus strengthening the need to develop large-scale systems using chemically defined media for ES cell production and/or controlled differentiation. In the present studies, a stirred culture system (i.e. spinner flask) was used to scale-up mouse ES (mES) cell expansion in serum-containing (DMEM/FBS) or serum-free medium, both supplemented with leukemia inhibitory factor (LIF), using either Cytodex 3 or Cultispher S microcarriers. After 8 days, maximal cell densities achieved were (1.9+/-0.1), (2.6+/-0.7) and 3.5x10(6)cells/mL for Cytodex 3 in DMEM/FBS, Cultispher S in DMEM/FBS and Cultispher S in serum-free cultures, respectively, with fold increases of 38+/-2, 50+/-15 and 70. Both microcarriers were suitable to sustain mES cell expansion, though the macroporous Cultispher S seemed to be advantageous in providing a more protective environment against shear stress forces, which harmful effects are exacerbated in serum-free conditions. Importantly, mES cells expanded under stirred conditions using serum-free medium retained their pluripotency and the ability to commit to the neural lineage.