rna-based-reprogramming-human-primary-fibroblasts-into-induced

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

circle-seq-for-interrogation-of-off-target-gene-editing

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of ...

University of Colorado School of Medicine, Anschutz Medical Campus

Recent advances in reprogramming somatic cells into induced pluripotent stem cells (iPSCs) offer the possibility of developing new therapeutic approaches for the treatment of a variety of diseases, including inherited skin disorders. While the ultimate goal is the use of iPSCs in the treatment of human diseases, extensive research is still required with preclinical mouse models before iPSC technology can be introduced into the clinic. Therefore, the methodology for the derivation of multipotent keratinocytes from mouse iPSCs is of particular importance since it may allow for the assessment of the feasibility of using iPSCs in the treatment of inherited skin disorders using mouse models which mimic these diseases. Here, we describe two alternative protocols for the efficient differentiation of mouse iPSCs into functional keratinocytes capable of reconstituting a normal stratified epidermis, hair follicles, and sebaceous glands when grafted onto mice. Each protocol results in a different yield and efficiency of keratinocyte derivation depending on the mouse genetic background used in the study. Both protocols employ applications of retinoic acid and bone-morphogenetic protein-4 and growth on collagen type IV-coated dishes to induce iPSC differentiation toward a keratinocyte lineage.

Generation of functional multipotent keratinocytes from mouse induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine; however, their potential clinical application is hampered by the low efficiency of somatic cell reprogramming. Here, we show that the synergistic activity of synthetic modified mRNAs encoding reprogramming factors and miRNA-367/302s delivered as mature miRNA mimics greatly enhances the reprogramming of human primary fibroblasts into iPSCs. This synergistic activity is dependent upon an optimal RNA transfection regimen and culturing conditions tailored specifically to human primary fibroblasts. As a result, we can now generate up to 4,019 iPSC colonies from only 500 starting human primary neonatal fibroblasts and reprogram up to 90.7% of individually plated cells, producing multiple sister colonies. This methodology consistently generates clinically relevant, integration-free iPSCs from a variety of human patient's fibroblasts under feeder-free conditions and can be applicable for the clinical translation of iPSCs and studying the biology of reprogramming.

High-efficiency RNA-based reprogramming of human primary fibroblasts.

Reprogramming a patient's somatic cells into induced pluripotent stem cells (iPSCs) holds great promise for disease modeling and the development of autologous cellular therapeutics. However, it remains challenging to consistently reprogram primary human cells, as they are frequently aged, diseased, or in low abundance. Here we present a modified highly efficient and clinically relevant RNA-based method for reprogramming disease-associated and other difficult-to-reprogram human primary fibroblast lines into iPSCs. We also describe optimizations that can be employed for consistent reprogramming of these difficult-to-reprogram cells. With the provided protocol, integration-free iPSC lines can be successfully generated from a small number of primary human fibroblasts in approximately 5-7 weeks.

Efficient RNA-Based Reprogramming of Disease-Associated Primary Human Fibroblasts into Induced Pluripotent Stem Cells.

The discovery of induced pluripotent stem cell (iPSC) technology has provided a versatile platform for basic science research and regenerative medicine. With the rise of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) systems and the ease at which they can be utilized for gene editing, creating genetically modified iPSCs has never been more advantageous for studying both organism development and potential clinical applications. However, to better understand the behavior and true therapeutic potential of iPSCs and iPSC-derived cells, a tool for labeling and monitoring these cells in vitro and in vivo is needed. Here, we describe a protocol that provides a straightforward method for introducing a stable, highly expressed fluorescent protein into iPSCs using the CRISPR/Cas9 system and a standardized donor vector. The approach involves the integration of the EGFP transgene into the transcriptionally active adeno-associated virus integration site 1 (AAVS1) locus through homology directed repair. The knockin of this transgene results in the generation of iPSC lines with constitutive expression of the EGFP protein that also persists in differentiated iPSCs. These EGFP-labeled iPSC lines are ideal for assessing iPSC differentiation in vitro and evaluating the distribution of iPSC-derived cells in vivo after transplantation into model animals.

Generation of an Induced Pluripotent Stem Cell Line with the Constitutive EGFP Reporter.

Differentiating human induced pluripotent stem cells (iPSCs) into multipotent mesenchymal stem/stromal cells (MSCs) offers a renewable source of therapeutically invaluable cells. However, the process of MSC derivation from iPSCs suffers from an undesirably low efficiency. In this chapter, we present an optimized procedure to produce MSCs from human iPSCs with a high efficiency. The protocol depends on the generation of embryoid bodies (EBs) and requires the treatment of EBs with transforming growth factor beta 1 (TGF-β1). The resulting MSCs can be purified based on the expression of CD73, CD105, and CD90 markers and expanded for multiple passages without losing their characteristics.

Differentiating Induced Pluripotent Stem Cells Toward Mesenchymal Stem/Stromal Cells.

Advancements in the molecular genetics of epidermolysis bullosa (EB) and ichthyosis, two rare inherited skin conditions, have enabled the identification of genetic variants that cause these diseases. Alongside technological advancements in genetic medicine, the identification of variants causal of these rare skin conditions has led to preclinical research and the clinical development of various in vivo and ex vivo gene and cell therapies for their treatment. Gene and cell therapies are considered to be the most advanced forms of personalized medicine, demonstrating safety and efficacy in numerous rare diseases. Although the orphan drug development boom has resulted in regulatory approval of multiple gene and cell therapies for various rare conditions, the application of these modalities to rare inherited skin conditions remains limited. Nonetheless, there are successful examples of both in vivo gene therapy- and ex vivo cell therapy-based approaches developed to treat EB and ichthyosis. This review highlights preclinical research and the clinical development of gene and cell therapies for multiple subtypes of these two devastating congenital skin conditions, including a gene therapy recently approved by the U.S. Food and Drug Administration for the treatment of recessive dystrophic EB.

Ganna Bilousova

Department of Dermatology,
Charles C. Gates Center for Regenerative Medicine,
Stem Cell Biobank and Disease Modeling Core,
Gates Institute

University of Colorado School of Medicine, Anschutz Medical Campus

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Ganna Bilousova

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Dermatology,Charles C. Gates Center for Regenerative Medicine,Stem Cell Biobank and Disease Modeling Core,Gates Institute

University of Colorado School of Medicine, Anschutz Medical Campus

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Department of Dermatology,
Charles C. Gates Center for Regenerative Medicine,
Stem Cell Biobank and Disease Modeling Core,
Gates Institute