The Impact of Acquired Genetic Abnormalities on the Clinical Translation of Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSC) are known to acquire chromosomal abnormalities, which range from point mutations to large copy number changes, including full chromosome aneuploidy. These aberrations have a wide-ranging influence on the state of cells, in both the undifferentiated and differentiated state. Currently, very little is known on how these abnormalities will impact the clinical translation of hPSC, and particularly their potential to prime cells for oncogenic transformation. A further complication is that many of these abnormalities exist in a mosaic state in culture, which complicates their detection with conventional karyotyping methods. In this review we discuss current knowledge on how these aberrations influence the cell state and how this may impact the future of research and the cells’ clinical potential.

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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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Base Editing Mediated Generation of Point Mutations Into Human Pluripotent Stem Cells for Modeling Disease

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.590581 ◽

2020 ◽

Vol 8 ◽

Author(s):

Tao Qi ◽

Fujian Wu ◽

Yuquan Xie ◽

Siqi Gao ◽

Miaomiao Li ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Point Mutations ◽

Human Pluripotent Stem Cells ◽

Base Editing

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Efficient GNE myopathy disease modeling with mutation specific phenotypes in human pluripotent stem cells by base editors

10.1101/2020.11.25.397711 ◽

2020 ◽

Author(s):

Ju-Chan Park ◽

Jumee Kim ◽

Hyun-Ki Jang ◽

Seung-Yeon Lee ◽

Keun-Tae Kim ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Point Mutations ◽

Human Pluripotent Stem Cells ◽

Drug Candidate ◽

Disease Models ◽

Target Cells ◽

Gne Myopathy ◽

Low Efficiency

AbstractIsogenic pairs of cell lines derived from human pluripotent stem cells (hPSCs) enable the precise assessment of mutation-specific phenotypes through differentiation to target cells, as this method of disease modeling excludes the bias of genetic variation. However, the extremely low efficiency of precise gene editing based on homology-directed repair (HDR) with Cas9 in hPSCs remains a technical hurdle for this approach. Herein, we took advantage of currently available base editors (BEs) in hPSCs to epitomize the isogenic disease model from hPSCs with a pathophysiological indicator. Using this method, we established 14 hPSCs that harbor point mutations on the GNE gene, including four different mutations found in GNE myopathy patients. Because BEs activated p53 to a lesser degree than Cas9, we observed a higher editing efficiency with BEs. Four different mutations in the epimerase or kinase domains of GNE revealed mutation-specific hyposialylation, which was closely correlated to pathological clinical phenotypes. These mutation-specific hyposialylation patterns were evident in GNE protein structure modeling. Furthermore, treatment with a drug candidate currently under clinical trials showed a mutation-specific drug response in GNE myopathy disease models. These data suggest that isogenic disease models from hPSCs using BEs could serve as a useful tool for mimicking the pathophysiology of GNE myopathy and for predicting drug responses.

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Pipeline for the Generation and Characterization of Transgenic Human Pluripotent Stem Cells Using the CRISPR/Cas9 Technology

Cells ◽

10.3390/cells9051312 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1312 ◽

Cited By ~ 2

Author(s):

Joffrey Mianné ◽

Chloé Bourguignon ◽

Chloé Nguyen Van ◽

Mathieu Fieldès ◽

Amel Nasri ◽

...

Keyword(s):

Stem Cells ◽

Cell Lines ◽

Pluripotent Stem Cells ◽

Genome Engineering ◽

Disease Modeling ◽

Mutant Cell ◽

Point Mutations ◽

Human Pluripotent Stem Cells ◽

Wide Range

Recent advances in genome engineering based on the CRISPR/Cas9 technology have revolutionized our ability to manipulate genomic DNA. Its use in human pluripotent stem cells (hPSCs) has allowed a wide range of mutant cell lines to be obtained at an unprecedented rate. The combination of these two groundbreaking technologies has tremendous potential, from disease modeling to stem cell-based therapies. However, the generation, screening and molecular characterization of these cell lines remain a cumbersome and multi-step endeavor. Here, we propose a pipeline of strategies to efficiently generate, sub-clone, and characterize CRISPR/Cas9-edited hPSC lines in the function of the introduced mutation (indels, point mutations, insertion of large constructs, deletions).

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Modeling Rett Syndrome with Human Pluripotent Stem Cells: Mechanistic Outcomes and Future Clinical Perspectives

International Journal of Molecular Sciences ◽

10.3390/ijms22073751 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3751

Author(s):

Ana Rita Gomes ◽

Tiago G. Fernandes ◽

Joaquim M.S. Cabral ◽

Maria Margarida Diogo

Keyword(s):

Stem Cells ◽

Rett Syndrome ◽

Pluripotent Stem Cells ◽

Neurodevelopmental Disorder ◽

Human Pluripotent Stem Cells ◽

Screening Tests ◽

Patient Specific ◽

Promising Alternative ◽

The Impact

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2). Among many different roles, MeCP2 has a high phenotypic impact during the different stages of brain development. Thus, it is essential to intensively investigate the function of MeCP2, and its regulated targets, to better understand the mechanisms of the disease and inspire the development of possible therapeutic strategies. Several animal models have greatly contributed to these studies, but more recently human pluripotent stem cells (hPSCs) have been providing a promising alternative for the study of RTT. The rapid evolution in the field of hPSC culture allowed first the development of 2D-based neuronal differentiation protocols, and more recently the generation of 3D human brain organoid models, a more complex approach that better recapitulates human neurodevelopment in vitro. Modeling RTT using these culture platforms, either with patient-specific human induced pluripotent stem cells (hiPSCs) or genetically-modified hPSCs, has certainly contributed to a better understanding of the onset of RTT and the disease phenotype, ultimately allowing the development of high throughput drugs screening tests for potential clinical translation. In this review, we first provide a brief summary of the main neurological features of RTT and the impact of MeCP2 mutations in the neuropathophysiology of this disease. Then, we provide a thorough revision of the more recent advances and future prospects of RTT modeling with human neural cells derived from hPSCs, obtained using both 2D and organoids culture systems, and its contribution for the current and future clinical trials for RTT.

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Toward clinical therapies using hematopoietic cells derived from human pluripotent stem cells

Blood ◽

10.1182/blood-2009-03-191304 ◽

2009 ◽

Vol 114 (17) ◽

pp. 3513-3523 ◽

Cited By ~ 109

Author(s):

Dan S. Kaufman

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Hematopoietic Cells ◽

Embryonic Stem ◽

Human Pluripotent Stem Cells ◽

Hematopoietic Cell ◽

Clinical Translation ◽

Current Status ◽

Cellular Mechanisms ◽

Hematopoietic Stem

Abstract Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide remarkable cellular platforms to better understand human hematopoiesis and to develop clinically applicable hematopoietic cell–based therapies. Over the past decade, hESCs have been used to characterize molecular and cellular mechanisms underpinning the differentiation of hematopoietic progenitors and mature, functional hematopoietic cells. These advances are now poised to lead to clinical translation of hESC- and iPSC-derived hematopoietic cells for novel therapies in the next few years. On the basis of areas of recent success, initial clinical use of hematopoietic cells derived from human pluripotent stem cells will probably be in the areas of transfusion therapies (erythrocytes and platelets) and immune therapies (natural killer cells). In contrast, efficient development and isolation of hematopoietic stem cells capable of long-term, multilineage engraftment still remains a significant challenge. Technical, safety, and regulatory concerns related to clinical applications of human PSCs must be appropriately addressed. However, proper consideration of these issues should facilitate and not inhibit clinical translation of new therapies. This review outlines the current status of hematopoietic cell development and what obstacles must be surmounted to bring hematopoietic cell therapies from human PSCs from “bench to bedside.”

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Generating microglia from human pluripotent stem cells: novel in vitro models for the study of neurodegeneration

Molecular Neurodegeneration ◽

10.1186/s13024-019-0347-z ◽

2019 ◽

Vol 14 (1) ◽

Cited By ~ 6

Author(s):

Anna M. Speicher ◽

Heinz Wiendl ◽

Sven G. Meuth ◽

Matthias Pawlowski

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Drug Testing ◽

Disease Modeling ◽

Current Knowledge ◽

Neurological Diseases ◽

Human Pluripotent Stem Cells ◽

Comprehensive Overview ◽

Human Microglia

AbstractMicroglia play an essential role for central nervous system (CNS) development and homeostasis and have been implicated in the onset, progression, and clearance of numerous diseases affecting the CNS. Previous in vitro research on human microglia was restricted to post-mortem brain tissue-derived microglia, with limited availability and lack of scalability. Recently, the first protocols for the generation of microglia from human pluripotent stem cells have become available, thus enabling the implementation of powerful platforms for disease modeling, drug testing, and studies on cell transplantation. Here we give a detailed and comprehensive overview of the protocols available for generating microglia from human pluripotent stem cells, highlighting the advantages, drawbacks, and operability and placing them into the context of current knowledge of human embryonic development. We review novel insights into microglia biology and the role of microglia in neurological diseases as drawn from the new methods and provide an outlook for future lines of research involving human pluripotent stem cell-derived microglia.

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