Gene Editing and Human Pluripotent Stem Cells: Tools for Advancing Diabetes Disease Modeling and Beta-Cell Development

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.

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Harnessing the Power of Induced Pluripotent Stem Cells and Gene Editing Technology: Therapeutic Implications in Hematological Malignancies

Cells ◽

10.3390/cells10102698 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2698

Author(s):

Ishnoor Sidhu ◽

Sonali P. Barwe ◽

Raju K. Pillai ◽

Anilkumar Gopalakrishnapillai

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Gene Editing ◽

Molecular Mechanisms ◽

Hematological Malignancies ◽

Disease Modeling ◽

Clonal Evolution ◽

Induced Pluripotent ◽

Ipsc Disease Modeling

In vitro modeling of hematological malignancies not only provides insights into the influence of genetic aberrations on cellular and molecular mechanisms involved in disease progression but also aids development and evaluation of therapeutic agents. Owing to their self-renewal and differentiation capacity, induced pluripotent stem cells (iPSCs) have emerged as a potential source of short in supply disease-specific human cells of the hematopoietic lineage. Patient-derived iPSCs can recapitulate the disease severity and spectrum of prognosis dictated by the genetic variation among patients and can be used for drug screening and studying clonal evolution. However, this approach lacks the ability to model the early phases of the disease leading to cancer. The advent of genetic editing technology has promoted the generation of precise isogenic iPSC disease models to address questions regarding the underlying genetic mechanism of disease initiation and progression. In this review, we discuss the use of iPSC disease modeling in hematological diseases, where there is lack of patient sample availability and/or difficulty of engraftment to generate animal models. Furthermore, we describe the power of combining iPSC and precise gene editing to elucidate the underlying mechanism of initiation and progression of various hematological malignancies. Finally, we discuss the power of iPSC disease modeling in developing and testing novel therapies in a high throughput setting.

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Human Pluripotent Stem Cells to Model Islet Defects in Diabetes

Frontiers in Endocrinology ◽

10.3389/fendo.2021.642152 ◽

2021 ◽

Vol 12 ◽

Author(s):

Diego Balboa ◽

Diepiriye G. Iworima ◽

Timothy J. Kieffer

Keyword(s):

Stem Cells ◽

Beta Cells ◽

Pluripotent Stem Cells ◽

Pancreatic Beta Cells ◽

Molecular Mechanisms ◽

Disease Modeling ◽

Human Pluripotent Stem Cells ◽

Pancreatic Endocrine Cells ◽

Onset Of Diabetes ◽

And Function

Diabetes mellitus is characterized by elevated levels of blood glucose and is ultimately caused by insufficient insulin production from pancreatic beta cells. Different research models have been utilized to unravel the molecular mechanisms leading to the onset of diabetes. The generation of pancreatic endocrine cells from human pluripotent stem cells constitutes an approach to study genetic defects leading to impaired beta cell development and function. Here, we review the recent progress in generating and characterizing functional stem cell-derived beta cells. We summarize the diabetes disease modeling possibilities that stem cells offer and the challenges that lie ahead to further improve these models.

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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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