Generation of 3D Skin Equivalents Fully Reconstituted from Human Induced Pluripotent Stem Cells (iPSCs)

Itoh, Munenari; Umegaki‐Arao, Noriko; Guo, Zongyou; Liu, Liang; Higgins, Claire A.; Christiano, Angela M.

doi:10.1371/journal.pone.0077673

Cited by 185 publications

(154 citation statements)

References 43 publications

(43 reference statements)

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“…S4). iPSC-derived keratinocytes and fibroblasts were generated as described previously (20,21). No differences were detected among normal, untreated DDEB, and gene-edited DDEB iPSC lines in the process of differentiation into keratinocytes and fibroblasts.…”

Section: Characterization Of Keratinocytes and Fibroblasts Derived Frmentioning

confidence: 99%

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Site-specific genome editing for correction of induced pluripotent stem cells derived from dominant dystrophic epidermolysis bullosa

Shinkuma

Guo

Christiano

2016

Proc. Natl. Acad. Sci. U.S.A.

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Genome editing with engineered site-specific endonucleases involves nonhomologous end-joining, leading to reading frame disruption. The approach is applicable to dominant negative disorders, which can be treated simply by knocking out the mutant allele, while leaving the normal allele intact. We applied this strategy to dominant dystrophic epidermolysis bullosa (DDEB), which is caused by a dominant negative mutation in the COL7A1 gene encoding type VII collagen (COL7). We performed genome editing with TALENs and CRISPR/Cas9 targeting the mutation, c.8068_8084delinsGA. We then cotransfected Cas9 and guide RNA expression vectors expressed with GFP and DsRed, respectively, into induced pluripotent stem cells (iPSCs) generated from DDEB fibroblasts. After sorting, 90% of the iPSCs were edited, and we selected four gene-edited iPSC lines for further study. These iPSCs were differentiated into keratinocytes and fibroblasts secreting COL7. RT-PCR and Western blot analyses revealed gene-edited COL7 with frameshift mutations degraded at the protein level. In addition, we confirmed that the gene-edited truncated COL7 could neither associate with normal COL7 nor undergo triple helix formation. Our data establish the feasibility of mutation site-specific genome editing in dominant negative disorders.CRISPR/Cas | TALENs | epidermolysis bullosa | gene editing | dominant negative effect

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Section: Characterization Of Keratinocytes and Fibroblasts Derived Frmentioning

confidence: 99%

“…iPSC-derived keratinocytes and fibroblasts were generated as described previously (20,21). These iPSCderived keratinocytes and fibroblasts were characterized by immunofluorescence studies as described previously (Table S3) (20,21).…”

mentioning

confidence: 99%

Site-specific genome editing for correction of induced pluripotent stem cells derived from dominant dystrophic epidermolysis bullosa

Shinkuma

Guo

Christiano

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…A good illustration is the work of Itoch et al [40] in which they elaborated a protocol of IPSCs differentiation into keratinocytes and dermal fibroblasts to be used in the treatment of recessive dystrophic epidermolysis bullosa. Besides, they created 3D equivalents of human skin consisting of keratinocytes and fibroblasts obtained from iPSCs only.…”

Section: A Cellular Componentmentioning

confidence: 99%

Skin Tissue-Engineering Constructs and Stem Cells Application for the Skin Equivalents Creation (Review)

Meleshina¹,

Bystrova²,

Rogovaya³

et al. 2017

Sovrem Tehnol Med

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Development and introduction of new biotechnological analogues (equivalents) of tissues and organs into clinical practice, such as human skin equivalents (SE), designed for temporal or permanent replacement of damaged or destroyed tissue, remains an urgent problem of regenerative medicine. Currently, full-thickness SE as well as separate skin layers, which include living cells of different types, are being created and investigated. Since the ideal skin substitutes have not been created, the efforts of researchers in many countries are aimed at solving this problem.In our review, we present a comparative analysis of existing SE, both commercial and those being at the stage of preclinical study, analyze their structure and feasibility of application for solving experimental and clinical tasks. Characteristics of the three main variants of SE have also been considered. Examples of stem cell application for creation of skin equivalents have been given. The main advantages of using stem cells as a cell component of skin equivalents have been described.

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“…iPSC models of organ and tissue development can then be monitored in real-time to identify any changes which may induce the onset of carcinogenesis. Recently, an in vitro model of skin was developed using iPSCs differentiated to keratinocytes and fibroblasts, providing an iPSC-generated in vitro model of a human organ [79] . Alternatively, iPSCs from normal somatic cells could also be manipulated to study carcinogenesis through overexpression or silencing of oncogenes, tumour suppressor genes and other factors thought to play a role in carcinogenesis, or alteration of the microenvironment.…”

Section: Modelling Carcinogenesis Using Non-malignant Cellsmentioning

confidence: 99%

Using induced pluripotent stem cells as a tool for modelling carcinogenesis

Curry

Moad

Robson

et al. 2015

WJSC

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Cancer is a highly heterogeneous group of diseases that despite improved treatments remain prevalent accounting for over 14 million new cases and 8.2 million deaths per year. Studies into the process of carcinogenesis are limited by lack of appropriate models for the development and pathogenesis of the disease based on human tissues. Primary culture of patient samples can help but is difficult to grow for a number of tissues. A potential opportunity to overcome these barriers is based on the landmark study by Yamanaka which demonstrated the ability of four factors; Oct4, Sox2, Klf4, and c-Myc to reprogram human somatic cells in to pluripotency. These cells were termed induced pluripotent stem cells (iPSCs) and display characteristic properties of embryonic stem cells. This technique has a wide range of potential uses including disease modelling, drug testing and transplantation studies. Interestingly iPSCs also share a number of characteristics with cancer cells including self-renewal and proliferation, expression of stem cell markers and altered metabolism. Recently, iPSCs have been generated from a number of human cancer cell lines and primary tumour samples from a range of cancers in an attempt to recapitulate the development of cancer and interrogate the underlying mechanisms involved. This review will outline the similarities between the reprogramming process and carcinogenesis, and how these similarities have been exploited to generate iPSC models for a number of cancers. Core tip: Human induced pluripotent stem cells (iPSCs) represent a novel method for studying the mechanisms of cancer development and progression. Recently, a number of studies have generated iPSCs from human cancer cells and cell lines, which can then be used as a model for carcinogenesis. This review outlines the similarities that exist between pluripotent and malignant cells and summarizes available studies that have generated iPSC models of cancer.

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Generation of 3D Skin Equivalents Fully Reconstituted from Human Induced Pluripotent Stem Cells (iPSCs)

Cited by 185 publications

References 43 publications

Site-specific genome editing for correction of induced pluripotent stem cells derived from dominant dystrophic epidermolysis bullosa

Site-specific genome editing for correction of induced pluripotent stem cells derived from dominant dystrophic epidermolysis bullosa

Skin Tissue-Engineering Constructs and Stem Cells Application for the Skin Equivalents Creation (Review)

Using induced pluripotent stem cells as a tool for modelling carcinogenesis

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