Serum replacement with albumin‐associated lipids prevents excess aggregation and enhances growth of induced pluripotent stem cells in suspension culture

Suspension culture systems are currently under investigation for the mass production of pluripotent stem (PS) cells for tissue engineering; however, the control of cell aggregation in suspension culture remains challenging. Existing methods to control aggregation such as microwell culture are difficult to scale up. To address this issue, in this study a novel method that incorporates the addition of KnockOut Serum Replacement (KSR) to the PS cell culture medium was described. The method regulated cellular aggregation and significantly improved cell growth (a 2‐ to 10‐fold increase) without any influence on pluripotency. In addition, albumin‐associated lipids as the major working ingredient of KSR responsible for this inhibition of aggregation were identified. This is one of the simplest methods described to date to control aggregation and requires only chemically synthesizable reagents. Thus, this method has the potential to simplify the mass production process of PS cells and thus lower their cost. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1009–1016, 2016     

Protection of human induced pluripotent stem cells against shear stress in suspension culture by Bingham plastic fluid

Suspension culture is an important method used in the industrial preparation of pluripotent stem cells (PSCs), for regenerative therapy and drug screening. Generally, a suspension culture requires agitation to keep PSC aggregates suspended and to promote mass transfer, but agitation also causes cell damage. In this study, we investigated the use of a Bingham plastic fluid, supplemented with a polysaccharide-based polymer, to preserve PSCs from cell damage in suspension culture. Rheometric analysis showed that the culture medium gained yield stress and became a Bingham plastic fluid, after supplementation with the polymer FP003. A growth/death analysis revealed that 2 days of aggregate formation and 2 days of suspension in the Bingham plastic medium improved cell growth and prevented cell death. After the initial aggregation step, whereas strong agitation (120 rpm) of a conventional culture medium resulted in massive cell death, in the Bingham plastic fluid we obtained the same growth as the normal culture with optimal agitation (90 rpm). This indicates that Bingham plastic fluid protected cells from shear stress in suspension culture and could be used to enhance their robustness when developing a large-scale.     

Polyvinyl alcohol modified polyvinylidene fluoride-graphene oxide scaffold promotes osteogenic differentiation potential of human induced pluripotent stem cells

Tissue engineering is fast becoming a key approach in bone medicine studies. Designing the ideally desirable combination of stem cells and scaffolds are at the hurt of efforts for producing implantable bone substitutes. Clinical application of stem cells could be associated with serious limitations, and engineering scaffolds that are able to imitate the important features of extracellular matrix is a major area of challenges within the field. In this study, electrospun scaffolds of polyvinylidene fluoride (PVDF), PVDF-graphene oxide (GO), PVDF-polyvinyl alcohol (PVA) and PVDF-PVA-GO were fabricated to study the osteogenic differentiation potential of human induced pluripotent stem cells (iPSCs) while cultured on fabricated scaffolds. Scanning electron microscopy study, viability assay, relative gene expression analysis, immunocytochemistry, alkaline phosphates activity, and calcium content assays confirmed that the osteogenesis rate of hiPSCs cultured on PVDF-PVA-Go is significantly higher than other scaffolds. Here, we showed that the biocompatible, nontoxic, flexible, piezoelectric, highly porous and interconnected three-dimensional structure of electrospun PVDF-PVA-Go scaffold in combination with hiPSCs (as the stem cells with significant advantageous in comparison to other types) makes them a highly promising scaffold-stem cell system for bone remodeling medicine. There was no evidence for the superiority of PVDF-GO or PVDF-PVA scaffold for osteogenesis, compared to each other; however both of them showed better potentials as to PVDF scaffold.     

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.     

Three-dimensional hydrogel culture conditions promote the differentiation of human induced pluripotent stem cells into hepatocytes

Background aims

Human induced pluripotent stem cells (hiPSCs) are becoming increasingly popular in research endeavors due to their potential for clinical application; however, such application is challenging due to limitations such as inferior function and low induction efficiency. In this study, we aimed to establish a three-dimensional (3D) culture condition to mimic the environment in which hepatogenesis occurs in vivo to enhance the differentiation of hiPSCs for large-scale culture and high throughput BAL application.

Developments in cell culture systems for human pluripotent stem cells

Human pluripotent stem cells (hPSCs) are important resources for cell-based therapies and pharmaceutical applications. In order to realize the potential of hPSCs, it is critical to develop suitable technologies required for specific applications. Most hPSC technologies depend on cell culture, and are critically influenced by culture medium composition, extracellular matrices, handling methods, and culture platforms. This review summarizes the major technological advances in hPSC culture, and highlights the opportunities and challenges in future therapeutic applications.     

Biobanking of human pluripotent stem cells in China

In recent years, significant progress has been made internationally in the development of human pluripotent stem cell (hPSC)‐derived products for serious and widespread disorders. Biobanking of the cellular starting materials is a crucial component in the delivery of safe and regulatory compliant cell therapies. In China, key players in these developments have been the recently launched National Stem Cell Resource Center (NSCRC) and its partner organizations in Guangzhou and Shanghai who together, have more than 600 hPSC lines formally recorded in the Chinese Ministry of Science and Technology''s stem cell registry. In addition, 47 of these hPSCs have also been registered with the hPSCreg project which means they are independently certified for use in European Commission funded research projects. The NSCRC are currently using their own cell lines to manufacture eight different cell types qualified for clinical use, that are being used in nine clinical studies for different indications. The Institute of Zoology at the Chinese Academy of Sciences (IOZ‐CAS) has worked with NSCRC to establish Chinese and international standards in stem cell research. IOZ‐CAS was also a founding partner in the International Stem Cell Banking Initiative which brings together key stem cell banks to agree minimum standards for the provision of pluripotent stem cells for research and clinical use. Here, we describe recent developments in China in the establishment of hPSCs for use in the manufacture of cell therapies and the significant national and international coordination which has now been established to promote the translation of Chinese hPSC‐based products into clinical use according to national and international standards.     

Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo

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Specific lectin biomarkers for isolation of human pluripotent stem cells identified through array-based glycomic analysis

Rapid and dependable methods for isolating human pluripotent stem cell (hPSC) populations are urgently needed for quality control in basic research and in cell-based therapy applications. Using lectin arrays, we analyzed glycoproteins extracted from 26 hPSC samples and 22 differentiated cell samples, and identified a small group of lectins with distinctive binding signatures that were sufficient to distinguish hPSCs from a variety of non-pluripotent cell types. These specific biomarkers were shared by all the 12 human embryonic stem cell and the 14 human induced pluripotent stem cell samples examined, regardless of the laboratory of origin, the culture conditions, the somatic cell type reprogrammed, or the reprogramming method used. We demonstrated a practical application of specific lectin binding by detecting hPSCs within a differentiated cell population with lectin-mediated staining followed by fluorescence microscopy and flow cytometry, and by enriching and purging viable hPSCs from mixed cell populations using lectin-mediated cell separation. Global gene expression analysis showed pluripotency-associated differential expression of specific fucosyltransferases and sialyltransferases, which may underlie these differences in protein glycosylation and lectin binding. Taken together, our results show that protein glycosylation differs considerably between pluripotent and non-pluripotent cells, and demonstrate that lectins may be used as biomarkers to monitor pluripotency in stem cell populations and for removal of viable hPSCs from mixed cell populations.     

Aneuploidy in pluripotent stem cells and implications for cancerous transformation

Owing to a unique set of attributes, human pluripotent stem cells (hPSCs) have emerged as a promising cell source for regenerative medicine, disease modeling and drug discovery. Assurance of genetic stability over long term maintenance of hPSCs is pivotal in this endeavor, but hPSCs can adapt to life in culture by acquiring non-random genetic changes that render them more robust and easier to grow. In separate studies between 12.5% and 34% of hPSC lines were found to acquire chromosome abnormalities over time, with the incidence increasing with passage number. The predominant genetic changes found in hPSC lines involve changes in chromosome number and structure (particularly of chromosomes 1, 12, 17 and 20), reminiscent of the changes observed in cancer cells. In this review, we summarize current knowledge on the causes and consequences of aneuploidy in hPSCs and highlight the potential links with genetic changes observed in human cancers and early embryos. We point to the need for comprehensive characterization of mechanisms underpinning both the acquisition of chromosomal abnormalities and selection pressures, which allow mutations to persist in hPSC cultures. Elucidation of these mechanisms will help to design culture conditions that minimize the appearance of aneuploid hPSCs. Moreover, aneuploidy in hPSCs may provide a unique platform to analyse the driving forces behind the genome evolution that may eventually lead to cancerous transformation.     

新物种诱导多能干细胞的研究进展

胚胎干细胞（embryonic stem cells,ESC）在人类遗传病学研究、疾病模型建立、器官再生以及动物物种改良和定向变异等方面的地位是其他类型的细胞不可取代的。但是,由于实验技术和体外培养条件的限制,除了小鼠、恒河猴和人之外,大鼠、猪、牛、羊等其他哺乳动物的ES细胞系被证明很难获得。先后有多个研究小组报道了他们利用新兴的诱导多能干细胞（induced pluripotent stem cells,iPS细胞）技术成功建立大鼠和猪的iPS细胞系的研究成果。迄今为止,这两个物种是在未成功建立ES细胞系之前利用iPS技术建立多能干细胞系的成功范例。这些研究对于那些还未建立ES细胞的物种建立多能干细胞系提供了一种新的方案,也将给这些物种的胚胎干细胞的建立、基因修饰动物的产生以及人类医疗事业的促进和发展带来新的希望。     

Heat shock protein 20 promotes sirtuin 1-dependent cell proliferation in induced pluripotent stem cells

BACKGROUNDHeat shock proteins (HSPs) are molecular chaperones that protect cells against cellular stresses or injury. However, it has been increasingly recognized that they also play crucial roles in regulating fundamental cellular processes. HSP20 has been implicated in cell proliferation, but conflicting studies have shown that it can either promote or suppress proliferation. The underlying mechanisms by which HSP20 regulates cell proliferation and pluripotency remain unexplored. While the effect of HSP20 on cell proliferation has been recognized, its role in inducing pluripotency in human-induced pluripotent stem cells (iPSCs) has not been addressed.AIMTo evaluate the efficacy of HSP20 overexpression in human iPSCs and evaluate the ability to promote cell proliferation. The purpose of this study was to investigate whether overexpression of HSP20 in iPSCs can increase pluripotency and regeneration.METHODSWe used iPSCs, which retain their potential for cell proliferation. HSP20 overexpression effectively enhanced cell proliferation and pluripotency. Overexpression of HSP20 in iPSCs was characterized by immunocytochemistry staining and real-time polymerase chain reaction. We also used cell culture, cell counting, western blotting, and flow cytometry analyses to validate HSP20 overexpression and its mechanism.RESULTSThis study demonstrated that overexpression of HSP20 can increase the pluripotency in iPSCs. Furthermore, by overexpressing HSP20 in iPSCs, we showed that HSP20 upregulated proliferation markers, induced pluripotent genes, and drove cell proliferation in a sirtuin 1 (SIRT1)-dependent manner. These data have practical applications in the field of stem cell-based therapies where the mass expansion of cells is needed to generate large quantities of stem cell-derived cells for transplantation purposes. CONCLUSIONWe found that the overexpression of HSP20 enhanced the proliferation of iPSCs in a SIRT1-dependent manner. Herein, we established the distinct crosstalk between HSP20 and SIRT1 in regulating cell proliferation and pluripotency. Our study provides novel insights into the mechanisms controlling cell proliferation that can potentially be exploited to improve the expansion and pluripotency of human iPSCs for cell transplantation therapies. These results suggest that iPSCs overexpressing HSP20 exert regenerative and proliferative effects and may have the potential to improve clinical outcomes.     

Large‐scale generation of megakaryocytes from human embryonic stem cells using transgene‐free and stepwise defined suspension culture conditions

ObjectivesEx vivo engineered production of megakaryocytes (MKs) and platelets (PLTs) from human pluripotent stem cells is an alternative approach to solve shortage of donor‐donated PLTs in clinics and to provide induced PLTs for transfusion. However, low production yields are observed and the generation of clinically applicable MKs and PLTs from human pluripotent stem cells without genetic modifications still needs to be improved.Materials and MethodsWe defined an optimal, stepwise and completely xeno‐free culture protocol for the generation of MKs from human embryonic stem cells (hESCs). To generate MKs from hESCs on a large scale, we improved the monolayer induction manner to define three‐dimensional (3D) and sphere‐like differentiation systems for MKs by using a special polystyrene CellSTACK culture chamber.ResultsThe 3D manufacturing system could efficiently generate large numbers of MKs from hESCs within 16‐18 days of continuous culturing. Each CellSTACK culture chamber could collect on an average 3.4 × 10⁸ CD41⁺ MKs after a three‐stage orderly induction process. MKs obtained from hESCs via 3D induction showed significant secretion of IL‐8, thrombospondin‐1 and MMP9. The induced cells derived from hESCs in our culture system were shown to have the characteristics of MKs as well as the function to form proPLTs and release PLTs. Furthermore, we generated clinically applicable MKs from clinical‐grade hESC lines and confirmed the biosafety of these cells.ConclusionsWe developed a simple, stepwise, 3D and completely xeno‐free/feeder‐free/transgene‐free induction system for the generation of MKs from hESCs. hESC‐derived MKs were shown to have typical MK characteristics and PLT formation ability. This study further enhances the clinical applications of MKs or PLTs derived from pluripotent stem cells.