direct-reprogramming-of-mouse-fibroblasts-into-melanocytes

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, ...

Jiangsu University

Affiliated Hospital of Jiangsu University

Jiangsu university

Induced pluripotent stem cells (iPSCs) are a promising melanocyte source as they propagate indefinitely and can be established from patients. However, the in vivo functions of human iPSC-derived melanocytes (hiMels) remain unknown. Here, we generated hiMels from vitiligo patients using a three-dimensional system with enhanced differentiation efficiency, which showed characteristics of human epidermal melanocytes with high sequence similarity and involved in multiple vitiligo-associated signaling pathways. A modified hair follicle reconstitution assay in vivo showed that MITFPAX3TYRP1 hiMels were localized in the mouse hair bulb and epidermis and produced melanin up to 7 weeks after transplantation, whereas MITFPAX3TYRP1 hiMelanocyte stem cells integrated into the bulge-subbulge regions. Overall, these data demonstrate the long-term functions of hiMels in vivo to reconstitute pigmented hair follicles and to integrate into normal regions for both mature melanocytes and melanocyte stem cells, providing an alternative source of personalized cellular therapy for depigmentation.

Therapeutic Potential of Patient iPSC-Derived iMelanocytes in Autologous Transplantation.

Induced pluripotent stem cells (iPSCs) show huge variations in their differentiation potential, even in the same condition. However, methods for predicting these differentiation tendencies, especially in the early stage of differentiation, are still scarce. This study aimed to establish a simple and practical system to predict the differentiation tendency of iPSC lines using embryoid bodies (EBs) with identified parameters in the early stage. We compared four human iPSC lines in terms of the morphology and maintenance of EBs and their gene expression levels of specific markers for three germ-layers. Furthermore, the differentiation potentials of these iPSC lines into melanocytes, which are ectoderm-derived cells, were also compared and correlated with the above parameters. The results showed that iPSC lines forming regular, smooth, and not cystic EBs, which could be maintained in culture for a relatively longer time, also expressed higher levels of ectoderm-specific markers and lower levels of mesoderm/endoderm markers. Additionally, these iPSC lines showed greater potential in melanocyte differentiation using EB-based protocol, and the induced melanocytes expressed melanocytic markers and presented characteristics that were similar to those of normal human melanocytes. By contrast, iPSC lines that formed cystic EBs with bright or dark cavities and expressed relatively lower levels of ectoderm-specific markers failed in the melanocyte differentiation. Collectively, the differentiation tendency of human iPSC lines may be predicted by specific parameters in the EB stage. The formation and maintenance of optimal EBs and the expression of germ layer-specific markers are particularly important and practical for the prediction assay in the early stage.

Early prediction of the differentiation potential during the formation of human iPSC-derived embryoid bodies.

The capability of human pluripotent stem cell (hPSC) lines to propagate indefinitely and differentiate into derivatives of three embryonic germ layers makes these cells be powerful tools for basic scientific research and promising agents for translational medicine. However, variations in differentiation tendency and efficiency as well as pluripotency maintenance necessitate the selection of hPSC lines for the intended applications to save time and cost. To screen the qualified cell lines and exclude problematic cell lines, their pluripotency must be confirmed initially by traditional methods such as teratoma formation or by high-throughput gene expression profiling assay. Additionally, their differentiation potential, particularly the lineage-specific differentiation propensities of hPSC lines, should be predicted in an early stage. As a complement to the teratoma assay, RNA sequencing data provide a quantitative estimate of the differentiation ability of hPSCs . Moreover, multiple scorecards have been developed based on selected gene sets for predicting the differentiation potential into three germ layers or the desired cell type many days before terminal differentiation. For clinical application of hPSCs, the malignant potential of the cells must also be evaluated. A combination of histologic examination of teratoma with quantitation of gene expression data derived from teratoma tissue provides safety-related predictive information by detecting immature teratomas, malignancy marker expression, and other parameters. Although various prediction methods are available, distinct limitations remain such as the discordance of results between different assays and requirement of a long time and high labor and cost, restricting their wide applications in routine studies. Therefore, simpler and more rapid detection assays with high specificity and sensitivity that can be used to monitor the status of hPSCs at any time and fewer targeted markers that are more specific for a given desired cell type are urgently needed.

Predicting differentiation potential of human pluripotent stem cells: Possibilities and challenges.

In the field of regenerative medicine, generating numerous transplantable functional cells in the laboratory setting on a large scale is a major challenge. However, the  maintenance and expansion of terminally differentiated cells are challenging because of the lack of specific environmental and intercellular signal stimulations, markedly hindering their therapeutic application. Remarkably, the generation of stem/progenitor cells or functional cells with effective proliferative potential is markedly in demand for disease modeling, cell-based transplantation, and drug discovery. Despite the potent genetic manipulation of transcription factors, integration-free chemically defined approaches for the conversion of somatic cell fate have garnered considerable attention in recent years. This review aims to summarize the progress thus far and discuss the advantages, limitations, and challenges of the impact of full chemicals on the stepwise reprogramming of pluripotency, direct lineage conversion, and direct lineage expansion on somatic cells. Owing to the current chemical-mediated induction, reprogrammed pluripotent stem cells with reproducibility difficulties, and direct lineage converted cells with marked functional deficiency, it is imperative to generate the desired cell types directly by chemically inducing their potent proliferation ability through a lineage-committed progenitor state, while upholding the maturation and engraftment capacity posttransplantation . Together with the comprehensive understanding of the mechanism of chemical drives, as well as the elucidation of specificity and commonalities, the precise manipulation of the expansion for diverse functional cell types could broaden the available cell sources and enhance the cellular function for clinical application in future.

Impelling force and current challenges by chemicals in somatic cell reprogramming and expansion beyond hepatocytes.

To solve the problem of liver transplantation donor insufficiency, an alternative cell transplantation therapy was investigated. We focused on amniotic epithelial cells (AECs) as a cell source because, unlike induced pluripotent stem cells, they are cost-effective and non-tumorigenic. The utilization of AECs in regenerative medicine, however, is in its infancy. A general profile for AECs has not been comprehensively analyzed. Moreover, no hepatic differentiation protocol for AECs has yet been established. To this end, we independently compiled human AEC libraries, purified amniotic stem cells (ASCs), and co-cultured them with mesenchymal stem cells (MSCs) and human umbilical vein endothelial cell (HUVECs) in a 3D system which induces functional hepatic organoids.

Enhanced hepatic differentiation in the subpopulation of human amniotic stem cells under 3D multicellular microenvironment.

A case of type I Kounis syndrome caused by double allergens.

Human induced pluripotent stem cells (hiPSCs) are invaluable resources for producing high-quality differentiated cells in unlimited quantities for both basic research and clinical use. They are particularly useful for studying human disease mechanisms  by making it possible to circumvent the ethical issues of human embryonic stem cell research. However, significant limitations exist when using conventional flat culturing methods especially concerning cell expansion, differentiation efficiency, stability maintenance and multicellular 3D structure establishment, differentiation prediction. Embryoid bodies (EBs), the multicellular aggregates spontaneously generated from iPSCs in the suspension system, might help to address these issues. Due to the unique microenvironment and cell communication in EB structure that a 2D culture system cannot achieve, EBs have been widely applied in hiPSC-derived differentiation and show significant advantages especially in scaling up culturing, differentiation efficiency enhancement,  simulation, and organoid establishment. EBs can potentially also be used in early prediction of iPSC differentiation capability. To improve the stability and feasibility of EB-mediated differentiation and generate high quality EBs, critical factors including iPSC pluripotency maintenance, generation of uniform morphology using micro-pattern 3D culture systems, proper cellular density inoculation, and EB size control are discussed on the basis of both published data and our own laboratory experiences. Collectively, the production of a large quantity of homogeneous EBs with high quality is important for the stability and feasibility of many PSCs related studies.

Inducing human induced pluripotent stem cell differentiation through embryoid bodies: A practical and stable approach.

The novel coronavirus disease 2019 (COVID-19) outbreak began in the city of Wuhan, whereupon it rapidly spread throughout China and subsequently across the world. Rapid transmission of COVID-19 has caused wide-spread panic. Many established medications have been used to treat the disease symptoms; however, no specific drugs or vaccines have been developed. Organoids derived from human induced pluripotent stem cells (iPSCs) may serve as suitable infection models for ex vivo mimicking of the viral life cycle and drug screening. Human iPSC-3D organoids, self-organised tissues with multiple cell environments, have a similar structure and function as real human organs; hence, these organoids allow greater viral infection efficiency, mimic the natural host-virus interactions, and are suitable for long-term experimentation. Here, we suggest the use of a functional human iPSC-organoid that could act as a reliable and feasible ex vivo infection model for investigation of the virus. This approach will provide much needed insight into the underlying molecular dynamics of COVID-19 for the development of novel treatment and prevention strategies.

A potential ex vivo infection model of human induced pluripotent stem cell-3D organoids beyond coronavirus disease 2019.

Human pluripotent stem cells (hPSCs) have the distinct advantage of being able to differentiate into cells of all three germ layers. Target cells or tissues derived from hPSCs have many uses such as drug screening, disease modeling, and transplantation therapy. There are currently a wide variety of differentiation methods available. However, most of the existing differentiation methods are unreliable, with uneven differentiation efficiency and poor reproducibility. At the same time, it is difficult to choose the optimal method when faced with so many differentiation schemes, and it is time-consuming and costly to explore a new differentiation approach. Thus, it is critical to design a robust and efficient method of differentiation. In this review article, we summarize a comprehensive approach in which hPSCs are differentiated into target cells or organoids including brain, liver, blood, melanocytes, and mesenchymal cells. This was accomplished by employing an embryoid body-based three-dimensional (3D) suspension culture system with multiple cells co-cultured. The method has high stable differentiation efficiency compared to the conventional 2D culture and can meet the requirements of clinical application. Additionally,  co-culture models might be able to constitute organoids that are highly similar or mimic human organs for potential organ transplantation in the future.

Practical choice for robust and efficient differentiation of human pluripotent stem cells.

Melanocytes, derived from neural crest cells, are involved in melanin production. This protocol describes a method to generate induced melanocytes (iMelanocytes) from human induced pluripotent stem cells (iPSCs) using a suspension culture system, which considerably improves the differentiation efficiency. The most critical parts of this protocol are the selection of a reliable iPSC line with strong potential to differentiate into melanocytes and their stemness maintenance. For complete information on the use and generation of this protocol, please refer to our  article, Liu el al. (2019).

Generation of Human iMelanocytes from Induced Pluripotent Stem Cells through a Suspension Culture System.

The Arabidopsis transcription factor NAC103 is up-regulated and its encoding protein is stabilized by ABA treatment, which positively regulates several ABA-responsive downstream genes during seed germination and seedlings growth. The Arabidopsis transcription factor NAC103 was previously found to be involved in endoplasmic reticulum (ER) stress and DNA damage responses. In this study, we report the new biological function of NAC103 in abscisic acid (ABA) response during seed germination and seedling growth in Arabidopsis. The expression of NAC103 was up-regulated and the NAC103 protein was stabilized by ABA treatment. Both the loss-of-function mutants of NAC103, created by targeted gene-editing, and the over-expression plants of NAC103 have no obvious germination-related phenotype under normal growth conditions. However, under exogenous ABA treatment conditions, the NAC103 mutants were less sensitive to ABA during seed germination; in contrast, the NAC103 over-expression plants were more sensitive to ABA during seed germination and young seedling growth. Further, NAC103 regulated several ABA-responsive downstream genes including MYB78, MYB3, PLP3, AMY1, and RGL2. These results demonstrate that NAC103 positively regulates ABA response in Arabidopsis.

NAC103, a NAC family transcription factor, regulates ABA response during seed germination and seedling growth in Arabidopsis.

As a standard clinical treatment, platelet transfusion has been employed to prevent hemorrhage in patients with thrombocytopenia or platelet dysfunctions. Platelets also show therapeutic potential for aiding liver regeneration and bone healing and regeneration and for treating dermatological conditions. However, the supply of platelets rarely meets the rising clinical demand. Other issues, including short shelf life, strict storage temperature, and allogeneic immunity caused by frequent platelet transfusions, have become serious challenges that require the development of high-yielding alternative sources of platelets. Human pluripotent stem cells (hPSCs) are an unlimited substitution source for regenerative medicine, and patient-derived iPSCs can provide novel research models to explore the pathogenesis of some diseases. Many studies have focused on establishing and modifying protocols for generating functional induced platelets (iPlatelets) from hPSCs. To reach high efficiency production and eliminate the exogenous antigens, media supplements and matrix have been optimized. In addition, the introduction of some critical transgenes, such as , , and , can also significantly increase hPSC-derived platelet production; however, this may pose some safety concerns. Furthermore, many novel culture systems have been developed to scale up the production of iPlatelets, including 2D flow systems, 3D rotary systems, and vertical reciprocal motion liquid culture bioreactors. The development of new gene-editing techniques, such as CRISPR/Cas9, can be used to solve allogeneic immunity of platelet transfusions by knocking out the expression of . Additionally, the functions of iPlatelets were also evaluated from multiple aspects, including but not limited to morphology, structure, cytoskeletal organization, granule content, DNA content, and gene expression. Although the production and functions of iPlatelets are close to meeting clinical application requirements in both quantity and quality, there is still a long way to go for their large-scale production and clinical application. Here, we summarize the diverse methods of platelet production and update the progresses of iPlatelets. Furthermore, we highlight recent advances in our understanding of key transcription factors or molecules that determine the platelet differentiation direction.

Article	Year
Therapeutic Potential of Patient iPSC-Derived iMelanocytes in Autologous Transplantation. Cell reports\| PubMed ID: 30970249	2019
Early prediction of the differentiation potential during the formation of human iPSC-derived embryoid bodies. Biochemical and biophysical research communications\| PubMed ID: 31248595	2019
Predicting differentiation potential of human pluripotent stem cells: Possibilities and challenges. World journal of stem cells\| PubMed ID: 31396366	2019
Impelling force and current challenges by chemicals in somatic cell reprogramming and expansion beyond hepatocytes. World journal of stem cells\| PubMed ID: 31616541	2019
Enhanced hepatic differentiation in the subpopulation of human amniotic stem cells under 3D multicellular microenvironment. World journal of stem cells\| PubMed ID: 31616545	2019
A case of type I Kounis syndrome caused by double allergens. Chinese medical journal\| PubMed ID: 32044817	2020
Inducing human induced pluripotent stem cell differentiation through embryoid bodies: A practical and stable approach. World journal of stem cells\| PubMed ID: 32110273	2020
A potential ex vivo infection model of human induced pluripotent stem cell-3D organoids beyond coronavirus disease 2019. Histology and histopathology\| PubMed ID: 32339250	2020
Practical choice for robust and efficient differentiation of human pluripotent stem cells. World journal of stem cells\| PubMed ID: 32952856	2020
Generation of Human iMelanocytes from Induced Pluripotent Stem Cells through a Suspension Culture System. STAR protocols\| PubMed ID: 33111066	2020
NAC103, a NAC family transcription factor, regulates ABA response during seed germination and seedling growth in Arabidopsis. Planta\| PubMed ID: 33130990	2020
The Opportunities and Challenges regarding Induced Platelets from Human Pluripotent Stem Cells. Stem cells international\| PubMed ID: 34054969	2021

Li-Ping Liu

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