scholarly journals Chemically defined and xeno-free culture condition for human extended pluripotent stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bei Liu ◽  
Shi Chen ◽  
Yaxing Xu ◽  
Yulin Lyu ◽  
Jinlin Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Culture Condition ◽  
Human Fibroblast ◽  
Culture System ◽  
Disease Modeling ◽  
Directed Differentiation

AbstractExtended pluripotent stem (EPS) cells have shown great applicative potentials in generating synthetic embryos, directed differentiation and disease modeling. However, the lack of a xeno-free culture condition has significantly limited their applications. Here, we report a chemically defined and xeno-free culture system for culturing and deriving human EPS cells in vitro. Xeno-free human EPS cells can be long-term and genetically stably maintained in vitro, as well as preserve their embryonic and extraembryonic developmental potentials. Furthermore, the xeno-free culturing system also permits efficient derivation of human EPS cells from human fibroblast through reprogramming. Our study could have broad utility in future applications of human EPS cells in biomedicine.

scholarly journals Directed Differentiation of Human Pluripotent Stem Cells toward Skeletal Myogenic Progenitors and Their Purification Using Surface Markers

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2746
Author(s):  
Nasa Xu ◽  
Jianbo Wu ◽  
Jose L. Ortiz-Vitali ◽  
Yong Li ◽  
Radbod Darabi
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Human Pluripotent Stem Cells ◽  
Directed Differentiation ◽  
Surface Markers ◽  
Gene Correction ◽  
Muscle Disorders

Advancements in reprogramming somatic cells into induced pluripotent stem cells (iPSCs) have provided a strong framework for in vitro disease modeling, gene correction and stem cell-based regenerative medicine. In cases of skeletal muscle disorders, iPSCs can be used for the generation of skeletal muscle progenitors to study disease mechanisms, or implementation for the treatment of muscle disorders. We have recently developed an improved directed differentiation method for the derivation of skeletal myogenic progenitors from hiPSCs. This method allows for a short-term (2 weeks) and efficient skeletal myogenic induction (45–65% of the cells) in human pluripotent stem cells (ESCs/iPSCs) using small molecules to induce mesoderm and subsequently myotomal progenitors, without the need for any gene integration or modification. After initial differentiation, skeletal myogenic progenitors can be purified from unwanted cells using surface markers (CD10+CD24−). These myogenic progenitors have been extensively characterized using in vitro gene expression/differentiation profiling as well as in vivo engraftment studies in dystrophic (mdx) and muscle injury (VML) rodent models and have been proven to be able to engraft and form mature myofibers as well as seeding muscle stem cells. The current protocol describes a detailed, step-by-step guide for this method and outlines important experimental details and troubleshooting points for its application in any human pluripotent stem cells.

scholarly journals Ascorbic Acid Can Promote the Generation and Expansion of Neuroepithelial-Like Stem Cells Derived from HiPS/ES Cells Under Chemically Defined Conditions Through Promoting Collagen Synthesis

2020 ◽  
Author(s):  
Rui Bai ◽  
Yun Chang ◽  
Amina Saleem ◽  
Fujian Wu ◽  
Lei Tian ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ascorbic Acid ◽  
Pluripotent Stem Cells ◽  
Culture System ◽  
Serum Free Medium ◽  
Cord Injury ◽  
Induction System ◽  
Defined Culture

Abstract Introduction: Spinal cord injury (SCI) is a neurological, medically incurable disorder. Human pluripotent stem cells (hPSCs) have the potential to generate neural stem/progenitor cells (NS/PCs) which hold promise in therapy for SCI by transplantation. In our study, we aimed to establish a chemically defined culture system by using serum-free medium and ascorbic acid (AA) to generation and expansion of long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) differentiated from hPSCs effectively and stably. Methods: We induce hESC/iPSC to neurospheres by using a newly established induction system in vitro in our study. And lt-NES cells derived from hESCs/iPSCs-neurospheres using two induction systems, including conventional N2 medium with gelatin-coated (coated) and N2+AA medium without pre-coated (AA) were characterized by reverse transcription-polymerase chain reaction (RT-PCR) analysis and immunocytochemistry staining. Subsequently, lt-NES cells were induced to neurons and the microelectrode array (MEA) recording system was used to evaluate the functionality of neurons differentiated from lt-NES cells. Moreover, the mechanism of AA-induced lt-NES cells was explored through RNA-seq and the use of inhibitors. Results: HESCs/iPSCs were efficiently induced to neurospheres by using a newly established induction system in vitro. And lt-NES cells derived from hESCs/iPSCs-neurospheres using two induction system (coated vs AA) both expressed neural pluripotency-associated genes PAX6, NESTIN, SOX1, SOX2. After long-term cultivation, we found that they both can maintain the long-term expansion for more than a dozen generations while maintaining neuropluripotency. Moreover, the lt-NES cells retain the ability to differentiate into general functional neurons that highly express β-tubulin. We also demonstrated that AA promotes the generation and long-term expansion of lt-NES cells by promoting collagen synthesis via the MEK-ERK1/2 pathways. Conclusions: Taken together, this new chemically defined culture system is stable and effective to generate and culture the lt-NES cells induced by hESCs/iPSCs using serum-free medium combined with ascorbic acid (AA). The lt-NES cells under this culture system can maintain the long-term expansion and neural pluripotency, with the potential to differentiate into functional neurons. Keywords: Spinal cord injury, Neurospheres, Ascorbic acid, lt-NES cells, Human pluripotent stem cells.

Generation of human hair-bearing skin organoids from stem cells

2020 ◽  
Author(s):  
Jiyoon Lee ◽  
Karl Koehler
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Human Hair ◽  
Culture System ◽  
Disease Modeling ◽  
Three Dimensional ◽  
Skin Tissue ◽  
Organoid Culture ◽  
Skin Biology

Abstract Skin is a complex and vulnerable tissue that it is challenging to reconstitute once damaged. Here, we describe a three-dimensional organoid culture system that can generate fully stratified skin with its appendages from human pluripotent stem cells. This in vitro-based skin organoid culture system will benefit investigations into basic skin biology and disease modeling, as well as translational efforts to reconstruct or regenerate skin tissue.

scholarly journals Generation of cortical neurons through large-scale expanding neuroepithelial stem cell from human pluripotent stem cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Shumei Zhao ◽  
Kui Duan ◽  
Zongyong Ai ◽  
Baohua Niu ◽  
Yanying Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Suspension Culture ◽  
Pluripotent Stem Cells ◽  
Cortical Neurons ◽  
Culture System ◽  
Large Scale ◽  
Disease Modeling ◽  
High Quality ◽  
Differentiation Potential

Abstract Background Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into cortical neurons for disease modeling and regenerative medicine. However, these procedures are hard to provide sufficient cells for their applications. Using a combination of small-molecules and growth factors, we previously identified one condition which can rapidly induce hPSCs into neuroepithelial stem cells (NESCs). Here, we developed a scalable suspension culture system, which largely yields high-quality NESC-spheres and subsequent cortical neurons. Methods The NESC medium was first optimized, and the suspension culture system was then enlarged from plates to stirred bioreactors for large-scale production of NESC-spheres by a stirring speed of 60 rpm. During the expansion, the quality of NESC-spheres was evaluated. The differentiation potential of NESC-spheres into cortical neurons was demonstrated by removing bFGF and two pathway inhibitors from the NESC medium. Cellular immunofluorescence staining, global transcriptome, and single-cell RNA sequencing analysis were used to identify the characteristics, identities, purities, or homogeneities of NESC-spheres or their differentiated cells, respectively. Results The optimized culture system is more conducive to large-scale suspension production of NESCs. These largely expanded NESC-spheres maintain unlimited self-renewal ability and NESC state by retaining their uniform sizes, high cell vitalities, and robust expansion abilities. After long-term expansion, NESC-spheres preserve high purity, homogeneity, and normal diploid karyotype. These expanded NESC-spheres on a large scale have strong differentiation potential and effectively produce mature cortical neurons. Conclusions We developed a serum-free, defined, and low-cost culture system for large-scale expansion of NESCs in stirred suspension bioreactors. The stable and controllable 3D system supports long-term expansion of high-quality and homogeneous NESC-spheres. These NESC-spheres can be used to efficiently give rise to cortical neurons for cell therapy, disease modeling, and drug screening in future.

scholarly journals Stability of Imprinting and Differentiation Capacity in Naïve Human Cells Induced by Chemical Inhibition of CDK8 and CDK19

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 876
Author(s):  
Raquel Bernad ◽  
Cian J. Lynch ◽  
Rocio G. Urdinguio ◽  
Camille Stephan-Otto Attolini ◽  
Mario F. Fraga ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cell ◽  
Normal Karyotype ◽  
Cell Types ◽  
Dna Demethylation ◽  
Starting State ◽  
Teratoma Formation

Pluripotent stem cells can be stabilized in vitro at different developmental states by the use of specific chemicals and soluble factors. The naïve and primed states are the best characterized pluripotency states. Naïve pluripotent stem cells (PSCs) correspond to the early pre-implantation blastocyst and, in mice, constitute the optimal starting state for subsequent developmental applications. However, the stabilization of human naïve PSCs remains challenging because, after short-term culture, most current methods result in karyotypic abnormalities, aberrant DNA methylation patterns, loss of imprinting and severely compromised developmental potency. We have recently developed a novel method to induce and stabilize naïve human PSCs that consists in the simple addition of a chemical inhibitor for the closely related CDK8 and CDK19 kinases (CDK8/19i). Long-term cultured CDK8/19i-naïve human PSCs preserve their normal karyotype and do not show widespread DNA demethylation. Here, we investigate the long-term stability of allele-specific methylation at imprinted loci and the differentiation potency of CDK8/19i-naïve human PSCs. We report that long-term cultured CDK8/19i-naïve human PSCs retain the imprinting profile of their parental primed cells, and imprints are further retained upon differentiation in the context of teratoma formation. We have also tested the capacity of long-term cultured CDK8/19i-naïve human PSCs to differentiate into primordial germ cell (PGC)-like cells (PGCLCs) and trophoblast stem cells (TSCs), two cell types that are accessible from the naïve state. Interestingly, long-term cultured CDK8/19i-naïve human PSCs differentiated into PGCLCs with a similar efficiency to their primed counterparts. Also, long-term cultured CDK8/19i-naïve human PSCs were able to differentiate into TSCs, a transition that was not possible for primed PSCs. We conclude that inhibition of CDK8/19 stabilizes human PSCs in a functional naïve state that preserves imprinting and potency over long-term culture.

scholarly journals The Promise of Human Induced Pluripotent Stem Cells in Dental Research

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Thekkeparambil Chandrabose Srijaya ◽  
Padmaja Jayaprasad Pradeep ◽  
Rosnah Binti Zain ◽  
Sabri Musa ◽  
Noor Hayaty Abu Kasim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Patient Specific ◽  
Dental Research ◽  
Cell Based Therapy ◽  
Induced Pluripotent ◽  
Disease Specific

Induced pluripotent stem cell-based therapy for treating genetic disorders has become an interesting field of research in recent years. However, there is a paucity of information regarding the applicability of induced pluripotent stem cells in dental research. Recent advances in the use of induced pluripotent stem cells have the potential for developing disease-specific iPSC linesin vitrofrom patients. Indeed, this has provided a perfect cell source for disease modeling and a better understanding of genetic aberrations, pathogenicity, and drug screening. In this paper, we will summarize the recent progress of the disease-specific iPSC development for various human diseases and try to evaluate the possibility of application of iPS technology in dentistry, including its capacity for reprogramming some genetic orodental diseases. In addition to the easy availability and suitability of dental stem cells, the approach of generating patient-specific pluripotent stem cells will undoubtedly benefit patients suffering from orodental disorders.

scholarly journals Directed differentiation and long-term maintenance of epicardial cells derived from human pluripotent stem cells under fully defined conditions

2017 ◽  
Vol 12 (9) ◽  
pp. 1890-1900 ◽  
Author(s):  
Xiaoping Bao ◽  
Xiaojun Lian ◽  
Tongcheng Qian ◽  
Vijesh J Bhute ◽  
Tianxiao Han ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Directed Differentiation ◽  
Epicardial Cells ◽  
Term Maintenance

scholarly journals Generation of star-shaped human induced astrocytes with a mature inflammatory phenotype for CNS disease modeling

2021 ◽  
Author(s):  
Dimitrios Voulgaris ◽  
Polyxeni Nikolakopoulou ◽  
Anna Herland
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Glutamate Transporter ◽  
Cns Disease ◽  
Human Neural Stem Cells ◽  
Induced Pluripotent ◽  
Prolonged Differentiation

Generating astrocytes from induced pluripotent stem cells has been hampered by either prolonged differentiation -spanning over two months -or by shorter protocols that generate immature astrocytes, devoid of salient inflammation-associated astrocytic traits pivotal for CNS neuropathological modeling. We directed human neural stem cells derived from induced pluripotent stem cells to astrocytic commitment and maturity by orchestrating an astrocytic-tuned culturing environment. In under 28 days, the generated cells express canonical and mature astrocytic markers, denoted by the expression of AQP4 and, remarkably, the expression and functionality of glutamate transporter EAAT2. We also show that this protocol generates astrocytes that encompass traits critical in CNS disease modeling, such as glutathione synthesis and secretion, upregulation of ICAM-1 and a cytokine secretion profile which is on par with primary astrocytes. This protocol generates a multifaceted astrocytic model suitable for CNS in vitro disease modeling and personalized medicine through brain-on-chip technologies.

scholarly journals Soft Substrate Maintains Proliferative and Adipogenic Differentiation Potential of human Mesenchymal Stem Cells on Long Term Expansion by Delaying Senescence

2018 ◽  
Author(s):  
Sanjay K. Kureel ◽  
Pankaj Mogha ◽  
Akshada Khadpekar ◽  
Vardhman Kumar ◽  
Rohit Joshi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Culture System ◽  
Human Mesenchymal Stem Cells ◽  
Population Doubling ◽  
Differentiation Potential ◽  
Lineage Differentiation ◽  
Poly Acrylamide

AbstractHuman mesenchymal stem cells (hMSCs), when cultured on tissue culture plate (TCP) for in vitro expansion, they spontaneously lose their proliferative capacity and multi-lineage differentiation potential. They also lose their distinct spindle morphology and become large and flat. After a certain number of population doubling, they enter into permanent cell cycle arrest, called senescence. This is a major roadblock for clinical use of hMSCs which demands large number of cells. A cell culture system is needed which can maintain the stemness of hMSCs over long term passages yet simple to use. In this study, we explore the role of substrate rigidity in maintaining stemness. hMSCs were serially passaged on TCP and 5 kPa poly-acrylamide gel for 20 population doubling. It was found that while on TCP, cell growth reached a plateau at cumulative population doubling (CPD) = 12.5, on 5 kPa gel, they continue to proliferate linearly till we monitored (CPD = 20). We also found that while on TCP, late passage MSCs lost their adipogenic potential, the same was maintained on soft gel. Cell surface markers related to MSCs were also unaltered. We demonstrated that this maintenance of stemness was correlated with delay in onset of senescence, which was confirmed by β-gal assay and by differential expression of vimentin, Lamin A and Lamin B. As preparation of poly-acrylamide gel is a simple, well established, and well standardized protocol, we believe that this system of cell expansion will be useful in therapeutic and research applications of hMSCs.One Sentence SummaryhMSCs retain their stemness when expanded in vitro on soft polyacrylamide gel coated with collagen by delaying senescence.Significance StatementFor clinical applications, mesenchymal stem cells (MSCs) are required in large numbers. As MSCs are available only in scarcity in vivo, to fulfill the need, extensive in vitro expansion is unavoidable. However, on expansion, they lose their replicative and multi-lineage differentiation potential and become senescent. A culture system that can maintain MSC stemness on long-term expansion, without compromising the stemness, is need of the hour. In this paper, we identified polyacrylamide (PAA) hydrogel of optimum stiffness that can be used to maintain stemness of MSCs during in vitro long term culture. Large quantity of MSCs thus grown can be used in regenerative medicine, cell therapy, and in treatment of inflammatory diseases.

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