Directed Differentiation of Human Pluripotent Stem Cells into Radial Glia and Astrocytes Bypasses Neurogenesis

AbstractDerivation of astrocytes from human pluripotent stem cells (hPSCs) is inefficient and cumbersome, impeding their use in biomedical research. Here, we developed a highly efficient chemically defined astrocyte differentiation strategy that overcomes current limitations. This approach largely bypasses neurogenesis, which otherwise precedes astrogliogenesis during brain development and in vitro experiments. hPSCs were first differentiated into radial glial cells (RGCs) exhibiting in vivo-like radial glia signatures. Activation of NOTCH and JAK/STAT pathways in bona fide RGCs resulted in direct astrogliogenesis confirmed by expression of various glial markers (NFIA, NFIB, SOX9, CD44, S100B, GFAP). Transcriptomic and genome-wide epigenetic analyses confirmed RGC-to-astrocyte differentiation and absence of neurogenesis. The morphological and functional identity of hPSC-derived astrocytes was confirmed by using an array of methods (e.g. electron microscopy, calcium imaging, co-culture with neurons, grafting into mouse brains). Lastly, the scalable protocol was adapted to a robotic platform and used to model Alexander disease. In conclusion, our findings uncover remarkable plasticity in neural lineage progression that can be exploited to manufacture large numbers of human hPSC-derived astrocytes for drug development and regenerative medicine.

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Synthetic probes for in vitro purification and in vivo tracking of hepatocytes derived from human pluripotent stem cells

Biomaterials ◽

10.1016/j.biomaterials.2019.119431 ◽

2019 ◽

Vol 222 ◽

pp. 119431 ◽

Cited By ~ 6

Author(s):

Ji Young Park ◽

Jiyou Han ◽

Hyo Sung Jung ◽

Gyunggyu Lee ◽

Hyo Jin Kim ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Human Pluripotent Stem Cells

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In Vitro Induction and In Vivo Engraftment of Lung Bud Tip Progenitor Cells Derived from Human Pluripotent Stem Cells

Stem Cell Reports ◽

10.1016/j.stemcr.2017.11.012 ◽

2018 ◽

Vol 10 (1) ◽

pp. 101-119 ◽

Cited By ~ 69

Author(s):

Alyssa J. Miller ◽

David R. Hill ◽

Melinda S. Nagy ◽

Yoshiro Aoki ◽

Briana R. Dye ◽

...

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Pluripotent Stem Cells ◽

Human Pluripotent Stem Cells ◽

In Vitro Induction

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Transcriptomically-guided mesendoderm induction of human pluripotent stem cells using a systematically defined culture scheme

10.1101/561068 ◽

2019 ◽

Author(s):

Richard L Carpenedo ◽

Sarah Y Kwon ◽

R Matthew Tanner ◽

Julien Yockell-Lelièvre ◽

Chandarong Choey ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Cell Biology ◽

Disease Modeling ◽

Human Pluripotent Stem Cells ◽

Endoderm Differentiation ◽

Prolonged Differentiation ◽

Defined Culture

SummaryHuman pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient, broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple, robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding (SCS) and BMP4 and Activin A (BA) treatment. Gene sets and gene ontology terms related to mesoderm and endoderm differentiation were enriched after 48 hours of BA treatment. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation. After prolonged differentiation in vitro or in vivo, BA pre-treatment resulted in higher mesoderm and endoderm levels at the expense of ectoderm formation. These data demonstrate that SCS with BA treatment is a powerful method for induction of mesendoderm that can be integrated into protocols for mesoderm and endoderm differentiation.

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Generation of hypoimmunogenic human pluripotent stem cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1902566116 ◽

2019 ◽

Vol 116 (21) ◽

pp. 10441-10446 ◽

Cited By ~ 43

Author(s):

Xiao Han ◽

Mengning Wang ◽

Songwei Duan ◽

Paul J. Franco ◽

Jennifer Hyoje-Ryu Kenty ◽

...

Keyword(s):

Stem Cells ◽

Cell Therapy ◽

Immune Responses ◽

Pluripotent Stem Cells ◽

Nk Cell ◽

Immune Surveillance ◽

Human Pluripotent Stem Cells ◽

Innate Immune

Polymorphic HLAs form the primary immune barrier to cell therapy. In addition, innate immune surveillance impacts cell engraftment, yet a strategy to control both, adaptive and innate immunity, is lacking. Here we employed multiplex genome editing to specifically ablate the expression of the highly polymorphic HLA-A/-B/-C and HLA class II in human pluripotent stem cells. Furthermore, to prevent innate immune rejection and further suppress adaptive immune responses, we expressed the immunomodulatory factors PD-L1, HLA-G, and the macrophage “don’t-eat me” signal CD47 from the AAVS1 safe harbor locus. Utilizing in vitro and in vivo immunoassays, we found that T cell responses were blunted. Moreover, NK cell killing and macrophage engulfment of our engineered cells were minimal. Our results describe an approach that effectively targets adaptive as well as innate immune responses and may therefore enable cell therapy on a broader scale.

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From Human Pluripotent Stem Cells to 3D Cardiac Microtissues: Progress, Applications and Challenges

Bioengineering ◽

10.3390/bioengineering7030092 ◽

2020 ◽

Vol 7 (3) ◽

pp. 92

Author(s):

Mariana A. Branco ◽

Joaquim M.S. Cabral ◽

Maria Margarida Diogo

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

High Throughput Screening ◽

Cardiac Development ◽

Human Pluripotent Stem Cells ◽

Cardiac Cells ◽

Cardiac Disorders ◽

The Impact

The knowledge acquired throughout the years concerning the in vivo regulation of cardiac development has promoted the establishment of directed differentiation protocols to obtain cardiomyocytes (CMs) and other cardiac cells from human pluripotent stem cells (hPSCs), which play a crucial role in the function and homeostasis of the heart. Among other developments in the field, the transition from homogeneous cultures of CMs to more complex multicellular cardiac microtissues (MTs) has increased the potential of these models for studying cardiac disorders in vitro and for clinically relevant applications such as drug screening and cardiotoxicity tests. This review addresses the state of the art of the generation of different cardiac cells from hPSCs and the impact of transitioning CM differentiation from 2D culture to a 3D environment. Additionally, current methods that may be employed to generate 3D cardiac MTs are reviewed and, finally, the adoption of these models for in vitro applications and their adaptation to medium- to high-throughput screening settings are also highlighted.

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Small molecule–driven direct conversion of human pluripotent stem cells into functional osteoblasts

Science Advances ◽

10.1126/sciadv.1600691 ◽

2016 ◽

Vol 2 (8) ◽

pp. e1600691 ◽

Cited By ~ 45

Author(s):

Heemin Kang ◽

Yu-Ru V. Shih ◽

Manando Nakasaki ◽

Harsha Kabra ◽

Shyni Varghese

Keyword(s):

Stem Cells ◽

Small Molecule ◽

Pluripotent Stem Cells ◽

Bone Matrix ◽

Induced Pluripotent Stem Cell ◽

Direct Conversion ◽

Human Pluripotent Stem Cells ◽

Tissue Specific

The abilities of human pluripotent stem cells (hPSCs) to proliferate without phenotypic alteration and to differentiate into tissue-specific progeny make them a promising cell source for regenerative medicine and development of physiologically relevant in vitro platforms. Despite this potential, efficient conversion of hPSCs into tissue-specific cells still remains a challenge. Herein, we report direct conversion of hPSCs into functional osteoblasts through the use of adenosine, a naturally occurring nucleoside in the human body. The hPSCs treated with adenosine not only expressed the molecular signatures of osteoblasts but also produced calcified bone matrix. Our findings show that the adenosine-mediated osteogenesis of hPSCs involved the adenosine A2bR. When implanted in vivo, using macroporous synthetic matrices, the human induced pluripotent stem cell (hiPSC)–derived donor cells participated in the repair of critical-sized bone defects through the formation of neobone tissue without teratoma formation. The newly formed bone tissues exhibited various attributes of the native tissue, including vascularization and bone resorption. To our knowledge, this is the first demonstration of adenosine-induced differentiation of hPSCs into functional osteoblasts and their subsequent use to regenerate bone tissues in vivo. This approach that uses a physiologically relevant single small molecule to generate hPSC-derived progenitor cells is highly appealing because of its simplicity, cost-effectiveness, scalability, and impact in cell manufacturing, all of which are decisive factors for successful translational applications of hPSCs.

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In vitro induction and in vivo engraftment of kidney organoids derived from human pluripotent stem cells

Experimental and Therapeutic Medicine ◽

10.3892/etm.2020.8844 ◽

2020 ◽

Vol 20 (2) ◽

pp. 1307-1314

Author(s):

Denglu Zhang ◽

Xiaohang Du ◽

Xufeng Zhang ◽

Kailin Li ◽

Feng Kong ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Human Pluripotent Stem Cells ◽

In Vitro Induction ◽

Kidney Organoids

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Controlling the switch from neurogenesis to pluripotency during marmoset monkey somatic cell reprogramming with self-replicating mRNAs and small molecules

10.1101/2020.05.21.107862 ◽

2020 ◽

Author(s):

Stoyan Petkov ◽

Ralf Dressel ◽

Ignacio Rodriguez-Polo ◽

Rüdiger Behr

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Disease Model ◽

Common Marmoset ◽

Animal Disease ◽

Neural Lineage ◽

Somatic Cell Reprogramming ◽

Fetal Fibroblasts

SUMMARYInduced pluripotent stem cells (iPSCs) hold enormous potential for the development of cell-based therapies for many currently incurable diseases. However, the safety and efficacy of potential iPSC-based treatments need to be verified in relevant animal disease models before their application in the clinic. Moreover, in order to reduce possible risks for the patients, it is necessary to use reprogramming approaches that ensure to the greatest extent possible the genomic integrity of the cells. Here, we report the derivation of iPSCs from common marmoset monkeys (Callithrix jacchus) using self-replicating mRNA vectors based on the Venezuelan equine encephalitis virus (VEE-mRNAs). By transfection of marmoset fetal fibroblasts with Tomato-modified VEE-mRNAs carrying the human OCT4, KLF4, SOX2, and c-MYC (VEE-OKS-iM-iTomato) and culture in medium supplemented with two small molecule inhibitors, we first established intermediate primary colonies with neural progenitor-like properties. In the second reprogramming step, we converted these colonies into transgene-free pluripotent stem cells by further culturing them with customized marmoset iPSC medium in feeder-free conditions. The resulting cell lines possess pluripotency characteristics, such as expression of various pluripotency markers, long-term self-renewal, stable karyotype, and ability to differentiate into derivatives of the three primary germ layers in vitro and in vivo. Our experiments reveal a novel paradigm for flexible reprogramming of somatic cells, where primary colonies obtained by a single VEE-mRNA transfection can be directed either towards the neural lineage or further reprogrammed to pluripotency. These results (i) will further enhance the role of the common marmoset as animal disease model for preclinical testing of iPSC-based therapies and (ii) establish an in vitro system to experimentally address developmental signal transduction pathways in primates.

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Neural Differentiation of Human Pluripotent Stem Cells for Nontherapeutic Applications: Toxicology, Pharmacology, andIn VitroDisease Modeling

Stem Cells International ◽

10.1155/2015/105172 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 15

Author(s):

May Shin Yap ◽

Kavitha R. Nathan ◽

Yin Yeo ◽

Lee Wei Lim ◽

Chit Laa Poh ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Blastocyst Stage ◽

Human Pluripotent Stem Cells ◽

Laboratory Animals ◽

Potential Therapeutic Application ◽

Human Cadavers ◽

Pharmacological Screening

Human pluripotent stem cells (hPSCs) derived from either blastocyst stage embryos (hESCs) or reprogrammed somatic cells (iPSCs) can provide an abundant source of human neuronal lineages that were previously sourced from human cadavers, abortuses, and discarded surgical waste. In addition to the well-known potential therapeutic application of these cells in regenerative medicine, these are also various promising nontherapeutic applications in toxicological and pharmacological screening of neuroactive compounds, as well as forin vitromodeling of neurodegenerative and neurodevelopmental disorders. Compared to alternative research models based on laboratory animals and immortalized cancer-derived human neural cell lines, neuronal cells differentiated from hPSCs possess the advantages of species specificity together with genetic and physiological normality, which could more closely recapitulatein vivoconditions within the human central nervous system. This review critically examines the various potential nontherapeutic applications of hPSC-derived neuronal lineages and gives a brief overview of differentiation protocols utilized to generate these cells from hESCs and iPSCs.

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Signaling Molecules Regulating Pancreatic Endocrine Development from Pluripotent Stem Cell Differentiation

International Journal of Molecular Sciences ◽

10.3390/ijms21165867 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5867

Author(s):

Hui Huang ◽

Taylor N. Bader ◽

Sha Jin

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Stem Cell Differentiation ◽

Signaling Molecules ◽

Human Pluripotent Stem Cells ◽

Diabetes Research ◽

Differentiation Pathways

Diabetes is one of the leading causes of death globally. Currently, the donor pancreas is the only source of human islets, placing extreme constraints on supply. Hence, it is imperative to develop renewable islets for diabetes research and treatment. To date, extensive efforts have been made to derive insulin-secreting cells from human pluripotent stem cells with substantial success. However, the in vitro generation of functional islet organoids remains a challenge due in part to our poor understanding of the signaling molecules indispensable for controlling differentiation pathways towards the self-assembly of functional islets from stem cells. Since this process relies on a variety of signaling molecules to guide the differentiation pathways, as well as the culture microenvironments that mimic in vivo physiological conditions, this review highlights extracellular matrix proteins, growth factors, signaling molecules, and microenvironments facilitating the generation of biologically functional pancreatic endocrine cells from human pluripotent stem cells. Signaling pathways involved in stepwise differentiation that guide the progression of stem cells into the endocrine lineage are also discussed. The development of protocols enabling the generation of islet organoids with hormone release capacities equivalent to native adult islets for clinical applications, disease modeling, and diabetes research are anticipated.

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