Ox‐LDL modifies the behaviour of bone marrow stem cells and impairs their endothelial differentiation via inhibition of Akt phosphorylation

This study was to investigate the effect of oxidized low‐density lipoprotein (ox‐LDL) on the behaviour of bone marrow stem cells and their endothelial differentiation as well as the underlying mechanisms. Adult rat bone marrow multipotent progenitor cells (MAPCs) were incubated with ox‐LDL for up to 2 weeks. Ox‐LDL treatment resulted in a time‐ and dose‐dependent reduction of MAPC population in culture through a combination of decreased cell proliferation and increased apoptosis. The expression of stem cell marker Oct‐4 was significantly suppressed in MAPCs by ox‐LDL in a dose‐ and time‐dependant manner. Endothelial differentiation of MAPCs was substantially inhibited by ox‐LDL with markedly decreased expression of endothelial markers vWF, Flk‐1 and CD31, as well as impaired in vitro vascular structure formation. Ox‐LDL‐induced apoptosis and inhibition of Oct‐4 expression, cell proliferation and endothelial differentiation of MAPCs were associated with significant inhibition of Akt phosphorylation. Akt overexpression in MAPCs transfected with a constitutively active Akt completely reversed the effects of ox‐LDL on MAPCs including enhanced apoptosis, decreased cell proliferation, suppressed Oct‐4 expression and endothelial differentiation as well as in vitro vascular structure formation. In conclusion, ox‐LDL promotes apoptosis and inhibits Oct‐4 expression and self‐renewal of MAPCs, and impairs their endothelial differentiation via suppression of Akt signalling.     

Stirred tank bioreactor culture combined with serum‐/xenogeneic‐free culture medium enables an efficient expansion of umbilical cord‐derived mesenchymal stem/stromal cells

Mesenchymal stem/stromal cells (MSC) are being widely explored as promising candidates for cell‐based therapies. Among the different human MSC origins exploited, umbilical cord represents an attractive and readily available source of MSC that involves a non‐invasive collection procedure. In order to achieve relevant cell numbers of human MSC for clinical applications, it is crucial to develop scalable culture systems that allow bioprocess control and monitoring, combined with the use of serum/xenogeneic (xeno)‐free culture media. In the present study, we firstly established a spinner flask culture system combining gelatin‐based Cultispher^®S microcarriers and xeno‐free culture medium for the expansion of umbilical cord matrix (UCM)‐derived MSC. This system enabled the production of 2.4 (±1.1) x10⁵ cells/mL (n = 4) after 5 days of culture, corresponding to a 5.3 (±1.6)‐fold increase in cell number. The established protocol was then implemented in a stirred‐tank bioreactor (800 mL working volume) (n = 3) yielding 115 million cells after 4 days. Upon expansion under stirred conditions, cells retained their differentiation ability and immunomodulatory potential. The development of a scalable microcarrier‐based stirred culture system, using xeno‐free culture medium that suits the intrinsic features of UCM‐derived MSC represents an important step towards a GMP compliant large‐scale production platform for these promising cell therapy candidates.     

Maintenance of pluripotency in mouse embryonic stem cells cultivated in stirred microcarrier cultures

The development of efficient and reproducible culture systems for embryonic stem (ES) cells is an essential pre‐requisite for regenerative medicine. Culture scale‐up ensuring maintenance of cell pluripotency is a central issue, because large amounts of pluripotent cells must be generated to warrant that differentiated cells deriving thereof are transplanted in great amounts and survive the procedure. This study aimed to develop a robust scalable cell expansion system, using a murine embryonic stem cell line that is feeder‐dependent and adapted to serum‐free medium, thus representing a more realistic model for human ES cells. We showed that high concentrations of murine ES cells can be obtained in stirred microcarrier‐based spinner cultures, with a 10‐fold concentration of cells per volume of medium and a 5‐fold greater cell concentration per surface area, as compared to static cultures. No differences in terms of pluripotency and differentiation capability were observed between cells grown in traditional static systems and cells that were replated onto the traditional system after being expanded on microcarriers in the stirred system. This was verified by morphological analyses, quantification of cells expressing important pluripotency markers (Oct‐4, SSEA‐1, and SOX2), karyotype profile, and the ability to form embryoid bodies with similar sizes, and maintaining their intrinsic ability to differentiate into all three germ layers. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

N‐acetylcysteine prevents oxidized low‐density lipoprotein‐induced reduction of MG53 and enhances MG53 protective effect on bone marrow stem cells

MG53 is an important membrane repair protein and partially protects bone marrow multipotent adult progenitor cells (MAPCs) against oxidized low‐density lipoprotein (ox‐LDL). The present study was to test the hypothesis that the limited protective effect of MG53 on MAPCs was due to ox‐LDL‐induced reduction of MG53. MAPCs were cultured with and without ox‐LDL (0‐20 μg/mL) for up to 48 hours with or without MG53 and antioxidant N‐acetylcysteine (NAC). Serum MG53 level was measured in ox‐LDL‐treated mice with or without NAC treatment. Ox‐LDL induced significant membrane damage and substantially impaired MAPC survival with selective inhibition of Akt phosphorylation. NAC treatment effectively prevented ox‐LDL‐induced reduction of Akt phosphorylation without protecting MAPCs against ox‐LDL. While having no effect on Akt phosphorylation, MG53 significantly decreased ox‐LDL‐induced membrane damage and partially improved the survival, proliferation and apoptosis of MAPCs in vitro. Ox‐LDL significantly decreased MG53 level in vitro and serum MG53 level in vivo without changing MG53 clearance. NAC treatment prevented ox‐LDL‐induced MG53 reduction both in vitro and in vivo. Combined NAC and MG53 treatment significantly improved MAPC survival against ox‐LDL. These data suggested that NAC enhanced the protective effect of MG53 on MAPCs against ox‐LDL through preventing ox‐LDL‐induced reduction of MG53.     

The role of albumin and PPAR‐α in differentiation‐dependent change of fatty acid profile during differentiation of mesenchymal stem cells to hepatocyte‐like cells

Differentiation of mesenchymal stem cells (MSCs) to hepatocyte‐like cells is associated with morphological and biological changes. In this study, the effect of hepatogenic differentiation on fatty acid profile and the expression of proliferator‐activated receptors‐α (PPAR‐α) have been studied. For this purpose, MSCs isolated from human umbilical cord were differentiated into hepatocyte‐like cells on selective culture media. The morphological and biochemical changes, PPAR‐α expression and reactive oxygen species (ROS) levels were studied during the differentiation process. Besides, the cells were processed to determine changes in fatty acid profile using gas chromatography analysis. The results showed that hepatic differentiation of the MSCs is associated with a decrease in major polyunsaturated fatty acids in mature hepatocytes, whereas there was an increase in the saturated fatty acid (SFA) levels during hepatocyte maturation. The differentiation‐dependent shift in the ratio of SFA/USFA was associated with changes in albumin and PPAR‐α expression, whereas changes in fatty acid profile were independent of ROS production and lipid peroxidation in differentiating cells. In conclusion, these data may suggest that hepatocyte formation during the stem cell differentiation is associated with a shift in the fatty acid profile that is probably a normal phenomenon in hepatogenic differentiation of the MSCs. Copyright © 2014 John Wiley & Sons, Ltd.     

Microenvironment Modulates Osteogenic Cell Lineage Commitment in Differentiated Embryonic Stem Cells

Background

Due to their self-renewal, embryonic stem cells (ESCs) are attractive cells for applications in regenerative medicine and tissue engineering. Although ESC differentiation has been used as a platform for generating bone in vitro and in vivo, the results have been unsatisfactory at best. It is possible that the traditional culture methods, which have been used, are not optimal and that other approaches must be explored.

Methodology/Principal Findings

ESCs were differentiated into osteoblast lineage using a micro-mass approach. In response to osteogenic differentiation medium, many cells underwent apoptosis, while others left the micro-mass, forming small aggregates in suspension. These aggregates were cultured in three different culture conditions (adhesion, static suspension, and stirred suspension), then examined for osteogenic potential in vitro and in vivo. In adhesion culture, ESCs primed to become osteoblasts recommitted to the adipocyte lineage in vitro. In a static suspension culture, resulting porous aggregates expressed osteoblasts markers and formed bone in vivo via intermembranous ossification. In a stirred suspension culture, resulting non-porous aggregates suppressed osteoblast differentiation in favor of expanding progenitor cells.

Conclusions/Significance

We demonstrate that microenvironment modulates cell fate and subsequent tissue formation during ESC differentiation. For effective tissue engineering using ESCs, it is important to develop optimized cell culture/differentiation conditions based upon the influence of microenvironment.     

Improved development of human embryonic stem cell-derived embryoid bodies by stirred vessel cultivation

Human embryonic stem cells (hESCs) represent an important resource for novel cell-based regenerative medical therapies. hESCs are known to differentiate into mature cells of defined lineages through the formation of embryoid bodies (EBs) which are amenable to suspension culture for several weeks. However, EBs derived from hESCs in standard static cultures are typically non-homogeneous, leading to inefficient cellular development. Here, we systematically compare the formation, growth, and differentiation capabilities of hESC-derived EBs in stirred and static suspension cultures. A 15-fold expansion in total number of EB-derived cells cultured for 21 days in a stirred flask was observed, compared to a fourfold expansion in static (non-stirred) cultures. Additionally, stirred vessel mediated cultures have a more homogeneous EB morphology and size. Importantly, the EBs cultivated in spinner flasks retained comparable ability to produce hematopoietic progenitor cells as those grown in static culture. These results demonstrate the decoupling between EB cultivation method and EB-derived cells' ability to form hematopoietic progenitors, and will allow for improved production of scalable quantities of hematopoietic cells or other differentiated cell lineages from hESCs in a controlled environment.     

Endothelial nitric oxide synthase is dynamically expressed during bone marrow stem cell differentiation into endothelial cells

This study was designed to investigate the developmental expression of endothelial nitric oxide synthase (eNOS) during stem cell differentiation into endothelial cells and to examine the functional status of the newly differentiated endothelial cells. Mouse adult multipotent progenitor cells (MAPCs) were used as the source of stem cells and were induced to differentiate into endothelial cells with vascular endothelial growth factor (VEGF) in serum-free medium. Expression of eNOS in the cells during differentiation was evaluated with real-time PCR, nitric oxide synthase (NOS) activity, and Western blot analysis. It was found that eNOS, but no other NOS, was present in undifferentiated MAPCs. eNOS expression disappeared in the cells immediately after induction of differentiation. However, eNOS expression reoccurred at day 7 during differentiation. Increasing eNOS mRNA, protein content, and activity were observed in the cells at days 14 and 21 during differentiation. The differentiated endothelial cells formed dense capillary networks on growth factor-reduced Matrigel. VEGF-stimulated phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2 occurred in these cells, which was inhibited by NOS inhibitor N(G)-nitro-L-arginine methyl ester. In conclusion, these data demonstrate that eNOS is present in MAPCs and is dynamically expressed during the differentiation of MAPCs into endothelial cells in vitro.     

Expansion strategies for human mesenchymal stromal cells culture under xeno‐free conditions

Choosing the culture system and culture medium used to produce cells are key steps toward a safe, scalable, and cost‐effective expansion bioprocess for cell therapy purposes. The use of AB human serum (AB HS) as an alternative xeno‐free supplement for mesenchymal stromal cells (MSC) cultivation has increasingly gained relevance due to safety and efficiency aspects. Here we have evaluated different scalable culture systems to produce a meaningful number of umbilical cord matrix‐derived MSC (UCM MSC) using AB HS for culture medium supplementation during expansion and cryopreservation to enable a xeno‐free bioprocess. UCM MSC were cultured in a scalable planar (compact 10‐layer flasks and roller bottles) and 3‐D microcarrier‐based culture systems (spinner flasks and stirred tank bioreactor). Ten layer flasks and roller bottles enabled the production of 2.6 ± 0.6 × 10⁴ and 1.4 ± 0.3 × 10⁴ cells/cm². UCM MSC‐based microcarrier expansion in the stirred conditions has enabled the production of higher cell densities (5.5–23.0 × 10⁴ cells/cm²) when compared to planar systems. Nevertheless, due to the moderate harvesting efficiency attained, (80% for spinner flasks and 46.6% for bioreactor) the total cell number recovered was lower than expected. Cells maintained the functional properties after expansion in all the culture systems evaluated. The cryopreservation of cells (using AB HS) was also successfully carried out. Establishing scalable xeno‐free expansion processes represents an important step toward a GMP compliant large‐scale production platform for MSC‐based clinical applications. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1358–1367, 2017     

Chemically controlled aggregation of pluripotent stem cells

Heterogeneity in pluripotent stem cell (PSC) aggregation leads to variability in mass transfer and signaling gradients between aggregates, which results in heterogeneous differentiation and therefore variability in product quality and yield. We have characterized a chemical‐based method to control aggregate size within a specific, tunable range with low heterogeneity, thereby reducing process variability in PSC expansion. This method enables controlled, scalable, stirred suspension‐based manufacturing of PSC cultures that are critical for the translation of regenerative medicine strategies to clinical products.     

Wnt3a Promotes Hippocampal Neurogenesis by Shortening Cell Cycle Duration of Neural Progenitor Cells

The effects of Wnt signaling on neural progenitor cells have been controversial. Activation of the canonical Wnt signaling pathway either promotes neural progenitor cell proliferation or accelerates their differentiation into postmitotic neurons. This study demonstrates that activation of the Wnt signaling pathway by itself induces neural progenitor cell proliferation but does not directly affect neuronal differentiation processes. To investigate whether Wnt signaling promotes expansion and/or differentiation of neural progenitor cells in the developing hippocampus, we prepared primary mouse hippocampal progenitors and treated them with Wnt3a in a chemically defined culture medium. Wnt3a increased the total number of cells, including the numbers of Ki67⁺ proliferating cells and Tuj1⁺ differentiated neurons. This result verified that Wnt3a promoted neural progenitor cell proliferation. Meanwhile, Wnt3a did not appear to actively enhance the neuronal differentiation process itself, because (1) the ratio of Tuj1⁺ cells to the total cells, and (2) the ratio of BrdU⁺ Tuj1⁺ cells to the total BrdU⁺ cells, were both comparable between cultures with or without Wnt3a. Indeed, Wnt3a caused no significant change in either cell survival or the proportion of symmetric and asymmetric cell divisions that directly affected neuron production. We finally demonstrated that the Wnt3a treatment simply shortened cell cycle duration of neural progenitor cells by 2.9 h. The accelerated cell cycle progression without affecting the ratio of symmetric/asymmetric cell divisions explains how Wnt signaling per se leads to the expansion of both proliferative cell population and differentiated neuronal cell population.     

Four stage liquid chromatographic selection of methionyl peptides for peptide-centric proteome analysis: the proteome of human multipotent adult progenitor cells

Serial application of strong cation-exchange and diagonal reversed-phase chromatography selecting methionyl peptides by stepwise shifting them from their reduced to their sulfoxide and sulfone forms generates a four-stage fractionation system, allowing high coverage analysis of complex proteome digests by LC-MALDI-MS/MS. Application to the proteome of a human multipotent adult progenitor cell line (MAPC) identified 2151 proteins with high confidence as on average four MS/MS-spectra were linked to each protein. Our dataset contains several novel, potential marker proteins that may be evaluated as affinity-anchors for isolating different adult stem cells in further studies. Furthermore, at least 2 tyrosine kinases that were previously linked to the self-renewal potential of stem cells were identified, validating the stemness of the analyzed cells. We also present data hinting at possible involvement of the ubiquitin/proteasome machinery in steering proliferation and/or differentiation of MAPC. Finally, following comparison of the MAPC proteome with proteomes of four human differentiated cell lines reveals differential usage of chromosomal information: compared to differentiated cells, MAPC do not appear to hold any preference for expressing genes located on specific chromosomes.     

Clonal expansion of hepatic progenitor cells and differentiation into hepatocyte-like cells

Hepatic progenitor cells (HPCs) in adult liver are promising for treatment of liver diseases. A biliary-derived HPC population in adult mice has been characterized by co-expression of stem cell marker Sry (sex determining region Y)-box 9 (SOX9) and biliary marker cytokeratin 7 (CK7). However, isolation of these HPCs in adult healthy liver without any selection procedures remains a big challenge in this field. Here, by establishing a simple and efficient method to isolate and expand the CK7⁺SOX9⁺ HPCs in vitro as clones, we acquired a stable and largely scalable cell source. The CK7⁺SOX9⁺ progenitor cells were then further induced to differentiate into hepatocyte-like cells with expression of mature hepatocyte markers albumin (Alb) and hepatocyte nuclear factor 4 alpha (HNF4α), both in vitro and in vivo in the presence of hepatocyte growth factor (HGF) and fibroblast growth factor 9 (FGF9). Furthermore, we found that the HPCs are highly responsive to transforming growth factor-beta (TGF-β) signals. Collectively, we identified and harvested a CK7⁺SOX9⁺ progenitor cell population from adult mouse liver by a simple and efficient approach. The exploration of this HPC population offers an alternative strategy of generating hepatocyte-like cells for cell-based therapies of acute and chronic liver disorders.     

Differentiation and Selection of Hepatocyte Precursors in Suspension Spheroid Culture of Transgenic Murine Embryonic Stem Cells

Embryonic stem cell-derived hepatocyte precursor cells represent a promising model for clinical transplantations to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology investigations. This study aimed to establish an in vitro culture system for scalable generation of hepatic progenitor cells. We used stable transgenic clones of murine embryonic stem cells possessing a reporter/selection vector, in which the enhanced green fluorescent protein- and puromycin N-acetyltransferase-coding genes are driven by a common alpha-fetoprotein gene promoter. This allowed for “live” monitoring and puromycin selection of the desired differentiating cell type possessing the activated alpha-fetoprotein gene. A rotary culture system was established, sequentially yielding initially partially selected hepatocyte lineage-committed cells, and finally, a highly purified cell population maintained as a dynamic suspension spheroid culture, which progressively developed the hepatic gene expression phenotype. The latter was confirmed by quantitative RT-PCR analysis, which showed a progressive up-regulation of hepatic genes during spheroid culture, indicating development of a mixed hepatocyte precursor-/fetal hepatocyte-like cell population. Adherent spheroids gave rise to advanced differentiated hepatocyte-like cells expressing hepatic proteins such as albumin, alpha-1-antitrypsin, cytokeratin 18, E-cadherin, and liver-specific organic anion transporter 1, as demonstrated by fluorescent immunostaining. A fraction of adherent cells was capable of glycogen storage and of reversible up-take of indocyanine green, demonstrating their hepatocyte-like functionality. Moreover, after transplantation of spheroids into the mouse liver, the spheroid-derived cells integrated into recipient. These results demonstrate that large-scale hepatocyte precursor-/hepatocyte-like cultures can be established for use in clinical trials, as well as in in vitro screening assays.     

Expansion of mouse sertoli cells on microcarriers

Background: Sertoli cells (SCs) have been described as the ‘nurse cells’ of the testis whose primary function is to provide essential growth factors and create an appropriate environment for development of other cells [for example, germinal and nerve stem cells (NSCs), used here]. However, the greatest challenge at present is that it is difficult to obtain sufficient SCs of normal physiological function for cell transplantation and biological medicine, largely due to traditional static culture parameter difficult to be monitored and scaled up. Objective: Operational stirred culture conditions for in vitro expansion and differentiation of SCs need to be optimized for large‐scale culture. Materials and methods: In this study, the culturing process for primary SC expansion and maintaining lack of differentiation was optimized for the first time, by using microcarrier bead technology in spinner flask culture. Effects of various feeding/refreshing regimes, stirring speeds, seed inoculum levels of SCs, and concentrations of microcarrier used for expansion of mouse SCs were also explored. In addition, pH, osmotic pressure and metabolic variables including consumption rates of glucose, glutamine, amino acids, and formation rates of lactic acid and ammonia, were investigated in culture. Results: After 6 days, maximal cell densities achieved were 4.6 × 10⁶ cells/ml for Cytodex‐1 in DMEM/FBS compared to 4.8 × 10⁵ cells/ml in static culture. Improved expansion was achieved using an inoculum of 1 × 10⁵ cells/ml and microcarrier concentration of 3 mg/ml at stirring speed of 30 rpm. Results indicated that medium replacement (50% changed everyday) resulted in supply of nutrients and removal of waste products inhibiting cell growth, that lead to maintenance of cultures in steady state for several days. These conditions favoured preservation of SCs in the undifferentiated state and significantly increased their physiological activity and trophic function, which were assessed by co‐culturing with NSCs and immunostaining. Conclusion: Data obtained in this study demonstrate the vast potential of this stirred culture system for efficient, reproducible and cost‐effective expansion of SCs in vitro. The system has advantages over static culture, which has major obstacles such as lower cell density, is time‐consuming and susceptible to contamination.