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Keratinocyte derivation procedure in defined conditions. See also Figure S4. A. Schematic drawing of the optimized keratinocyte differentiation protocol. B. Cell morphology changes in the differentiation process. Scale bar,100µm. C. Stagewise keratinocyte gene expression by RT-qPCR. The results were normalized to hESC (D0); Data are representative of 3 independent experiments. D. Immunostaining of TP63 (red), KRT14 (green), KRT1 (green) and KRT10 (green) on day 24 and day 32 of differentiation. Scale bar, 50µm. E. Flow cytometry analysis of KRT14 (left panel) and KRT1 (right panel) on Day 28 of differentiation. Blue peak represents undifferentiated hESCs; pink peak represents keratinocyte. F. Keratinocyte differentiation from multiple hESC (H1, H9) and hiPSC (ND1-4, NL-1, NL-4) lines. Gene expression was analyzed on day 20 of differentiation and compared with HaCaT keratinocyte cell line, primary human foreskin keratinocytes (HFK-1, HFK-2) and undifferentiated hESCs (H1). The results were normalized to hESC (H1); Data are representative of 3 independent experiments.
Source publication
Keratinocyte is the predominant cell type in the epidermis of skin, and it provides the protective barrier function for the body. Various signaling pathways have been implicated in keratinocyte differentiation in animal models; However, their temporal regulation and interactions are still to be explored in pluripotent stem cell models. In this repo...
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... on the above findings, we were able to differentiate hESCs to keratinocyte lineage, and we then cultured the cells further in a defined medium that was formulated according to previous reports by others [36]. Finally, we established a procedure to differentiate hESCs toward keratinocytes through multiple stages ( Figure 4A). In the process, we observed significant changes in the cellular morphology ( Figure 4B). ...
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... we established a procedure to differentiate hESCs toward keratinocytes through multiple stages ( Figure 4A). In the process, we observed significant changes in the cellular morphology ( Figure 4B). The epidermal marker gene expression changes were consistent with differentiation stages ( Figure 4C). ...
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... the process, we observed significant changes in the cellular morphology ( Figure 4B). The epidermal marker gene expression changes were consistent with differentiation stages ( Figure 4C). After the cells were differentiated for more than 20 days, most cells expressed keratinocyte markers, including TP63, KRT14, KRT10 and KRT1 ( Figure 4D and S4D). ...
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... epidermal marker gene expression changes were consistent with differentiation stages ( Figure 4C). After the cells were differentiated for more than 20 days, most cells expressed keratinocyte markers, including TP63, KRT14, KRT10 and KRT1 ( Figure 4D and S4D). This observation was consistent with flow cytometry results that >95% of the cells were positive for KRT14 and ＞ 85% of cells express maturation marker KRT1 ( Figure 4E). ...
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... the cells were differentiated for more than 20 days, most cells expressed keratinocyte markers, including TP63, KRT14, KRT10 and KRT1 ( Figure 4D and S4D). This observation was consistent with flow cytometry results that >95% of the cells were positive for KRT14 and ＞ 85% of cells express maturation marker KRT1 ( Figure 4E). This differentiation process can also be carried out in fully chemically defined culture conditions, in the absence of Matrigel and albumin. ...
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... differentiation process can also be carried out in fully chemically defined culture conditions, in the absence of Matrigel and albumin. We showed that keratinocytes were generated on recombinant vitronectin-coated surface ( Figure S4A), and in albumin-free conditions ( Figure S4B). The keratinocytes generated have high expansion capacity ( Figure S4C) and can be passaged 8-10 times in vitro. ...
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... differentiation process can also be carried out in fully chemically defined culture conditions, in the absence of Matrigel and albumin. We showed that keratinocytes were generated on recombinant vitronectin-coated surface ( Figure S4A), and in albumin-free conditions ( Figure S4B). The keratinocytes generated have high expansion capacity ( Figure S4C) and can be passaged 8-10 times in vitro. ...
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... showed that keratinocytes were generated on recombinant vitronectin-coated surface ( Figure S4A), and in albumin-free conditions ( Figure S4B). The keratinocytes generated have high expansion capacity ( Figure S4C) and can be passaged 8-10 times in vitro. ...
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... keratinocyte differentiation procedure was then applied to multiple hPSC lines, including hESC (H1, H9) and hiPSC (NL-1, NL-4, ND1-4) lines. Most cell lines showed positive expression of keratinocyte genes, and the levels were comparable to the immortalized keratinocyte cell line HaCaT and primary human keratinocyte cell lines ( Figure 4F). We also noticed that different cell lines vary in the level of marker gene expression. ...
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... further evaluate the potential application of our hPSC-derived keratinocytes in clinical transplantation, we tested the in vivo survival and incorporation of hESC-derived keratinocytes in a mouse model. A GFP-labeled hESC reporter cell line was produced, and cells were differentiated to keratinocytes following the protocol above ( Figure 4A). Cells were collected on day 20 and transplanted into excisional wounds on immunodeficient mice by injection and topical application [38]. ...
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... we used TP63, but not KRT18, as the key marker to track the epidermal differentiation in this study. Third, we noticed that basal and suprabasal marker genes are dynamically expressed in our differentiation processes (Figure 4). Our results suggest that basal and suprabasal genes could be expressed in the same progenitor cells. ...
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We sought to elucidate how and when the ocular surface ectoderm commits to its differentiation into the corneal epithelium in eye development from human induced pluripotent stem cells (hiPSCs) under the influence of WNT signaling and the actions of BMP4. These signals are key drivers ocular surface ectodermal cell fate determination. It was discove...
Citations
... While the TGFβ/Activin/Nodal pathway is known to induce mesoderm and endoderm fate [7]. Therefore, activating BMP signaling and inhibiting TGFβ signaling by BMP4 and SB431542 were used to induce NNE from human pluripotent stem cells [8]. It was reported that early retinoic acid (RA) application led to the emergence of unidentified non-neural cell fates [9]. ...
Human-induced pluripotent stem cells (hiPSCs) offer a promising source for generating dental epithelial (DE) cells. Whereas the existing differentiation protocols were time-consuming and relied heavily on growth factors, herein, we developed a three-step protocol to convert hiPSCs into DE cells in 8 days. In the first phase, hiPSCs were differentiated into non-neural ectoderm using SU5402 (an FGF signaling inhibitor). The second phase involved differentiating non-neural ectoderm into pan-placodal ectoderm and simultaneously inducing the formation of oral ectoderm (OE) using LDN193189 (a BMP signaling inhibitor) and purmorphamine (a SHH signaling activator). In the final phase, OE cells were differentiated into DE through the application of Purmorphamine, XAV939 (a WNT signaling inhibitor), and BMP4. qRT-PCR and immunostaining were performed to examine the expression of lineage-specific markers. ARS staining was performed to evaluate the formation of the mineralization nodule. The expression of PITX2, SP6, and AMBN, the emergence of mineralization nodules, and the enhanced expression of AMBN and AMELX in spheroid culture implied the generation of DE cells. This study delineates the developmental signaling pathways and uses small molecules to streamline the induction of hiPSCs into DE cells. Our findings present a simplified and quicker method for generating DE cells, contributing valuable insights for dental regeneration and dental disease research.
... Keratinocytes are the predominant cell type in the epidermis of the skin and provide a protective barrier for the body (Zhong et al., 2020). Numerous signaling pathways have been implicated in keratinocyte differentiation, including the TGF-β, NOTCH, Hedgehog, and Hippo pathways (Zhong et al., 2020). ...
... Keratinocytes are the predominant cell type in the epidermis of the skin and provide a protective barrier for the body (Zhong et al., 2020). Numerous signaling pathways have been implicated in keratinocyte differentiation, including the TGF-β, NOTCH, Hedgehog, and Hippo pathways (Zhong et al., 2020). TGF-β signaling is particularly important in the differentiation and keratinization processes of epidermal cells (Cheng et al., 2016). ...
... Filaggrin, loricrin, Fig. 3 The most important molecular pathways involved in the differentiation of keratinocytes include cell receptors and a number of main proteins. In this figure, to avoid becoming more complicated, the dis-play of molecular pathways involved in apoptosis, growth and migration of keratinocytes has been omitted [16,30, and caspase-14 activation are involved in terminal keratinocyte differentiation; while transglutaminase and involucrin are indicators that start at the granular layer [155]. ...
... Zhong et al. showed that human embryonic stem cellderived keratinocytes survived and matured when transplanted into mice, showing the potential for clinical applications [30]. ...
To improve wound healing or treatment of other skin diseases, and provide model cells for skin biology studies, in vitro differentiation of stem cells into keratinocyte-like cells (KLCs) is very desirable in regenerative medicine. This study examined the most recent advancements in in vitro differentiation of stem cells into KLCs, the effect of biofactors, procedures, and preparation for upcoming clinical cases. A range of stem cells with different origins could be differentiated into KLCs under appropriate conditions. The most effective ways of stem cell differentiation into keratinocytes were found to include the co-culture with primary epithelial cells and keratinocytes, and a cocktail of growth factors, cytokines, and small molecules. KLCs should also be supported by biomaterials for the extracellular matrix (ECM), which replicate the composition and functionality of the in vivo extracellular matrix (ECM) and, thus, support their phenotypic and functional characteristics. The detailed efficient characterization of different factors, and their combinations, could make it possible to find the significant inducers for stem cell differentiation into epidermal lineage. Moreover, it allows the development of chemically known media for directing multi-step differentiation procedures.
In conclusion, the differentiation of stem cells to KLCs is feasible and KLCs were used in experimental, preclinical, and clinical trials. However, the translation of KLCs from in vitro investigational system to clinically valuable cells is challenging and extremely slow.
Graphical Abstract
... During the 17 days of tissue maturation, barrier integrity was monitored by TEER and showed a progressive tightness increase over time (Fig. 5b). After 14 days of co-culture required for keratinocyte maturation 47,48 (experiment day 17), barrier permeability was compared between TEER (Fig. 5c) and the diffusion of 0.4 kDa low molecular weight dextran from top to bottom (Fig. 5d). We noticed a high correlation between both TEER and dextran measurements (Fig. 5b-c). ...
The development of new therapies is hampered by the lack of predictive, and patient-relevant in vitro models. Organ-on-chip (OOC) technologies can potentially recreate physiological features and hold great promise for tissue and disease modeling. However, the non-standardized design of these chips and perfusion control systems has been a barrier to quantitative high-throughput screening (HTS).
Here we present a scalable OOC microfluidic platform for applied kinetic in vitro assays (AKITA) that is applicable for high, medium, and low throughput. Its standard 96-well plate and 384-well plate layouts ensure compatibility with existing laboratory workflows and high-throughput data collection and analysis tools. The AKITA plate is optimized for the modeling of vascularized biological barriers, primarily the blood-brain barrier, skin, and lung, with precise flow control on a custom rocker. The integration of trans-epithelial electrical resistance (TEER) sensors allows rapid and repeated monitoring of barrier integrity over long time periods.
Together with automated confocal imaging and compound permeability testing analyses, we demonstrate the flexibility of the AKITA platform for establishing human-relevant models for preclinical drug and precision medicine's efficacy, toxicity, and permeability under physiological conditions.
... TGFβ signaling mediates mesoderm development, and inhibition of TGFβ signaling is necessary for the fate switch from fibroblasts or astrocytes to other cells [40,41]. The activation of WNT signaling and BMP signaling and the inhibition of TGFβ signaling have been found to increase CK18, CK14 and CK10 [42]. Interestingly, our results also confirmed the differences in the above signaling pathways between fibroblasts and pSGCs, thus suggesting that targeted regulation of these pathways may enhance the efficiency of SG reprogramming. ...
Background
Large skin defects severely disrupt the overall skin structure and can irreversibly damage sweat glands (SG), thus impairing the skin’s physiological function. This study aims to develop a stepwise reprogramming strategy to convert fibroblasts into SG lineages, which may provide a promising method to obtain desirable cell types for the functional repair and regeneration of damaged skin.
Methods
The expression of the SG markers cytokeratin 5 (CK5), cytokeratin 10 (CK10), cytokeratin 18 (CK18), carcino-embryonic antigen (CEA), aquaporin 5 (AQP5) and α-smooth muscle actin (α-SMA) was assessed with quantitative PCR (qPCR), immunofluorescence and flow cytometry. Calcium activity analysis was conducted to test the function of induced SG-like cells (iSGCs). Mouse xenograft models were also used to evaluate the in vivo regeneration of iSGCs. BALB/c nude mice were randomly divided into a normal group, SGM treatment group and iSGC transplantation group. Immunocytochemical analyses and starch-iodine sweat tests were used to confirm the in vivo regeneration of iSGCs.
Results
EDA overexpression drove HDF conversion into iSGCs in SG culture medium (SGM). qPCR indicated significantly increased mRNA levels of the SG markers CK5, CK18 and CEA in iSGCs, and flow cytometry data demonstrated (4.18 ± 0.04)% of iSGCs were CK5 positive and (4.36 ± 0.25)% of iSGCs were CK18 positive. The addition of chemical cocktails greatly accelerated the SG fate program. qPCR results revealed significantly increased mRNA expression of CK5, CK18 and CEA in iSGCs, as well as activation of the duct marker CK10 and luminal functional marker AQP5. Flow cytometry indicated, after the treatment of chemical cocktails, (23.05 ± 2.49)% of iSGCs expressed CK5 ⁺ and (55.79 ± 3.18)% of iSGCs expressed CK18 ⁺ , respectively. Calcium activity analysis indicated that the reactivity of iSGCs to acetylcholine was close to that of primary SG cells [(60.79 ± 7.71)% vs. (70.59 ± 0.34)%, ns]. In vivo transplantation experiments showed approximately (5.2 ± 1.1)% of the mice were sweat test positive, and the histological analysis results indicated that regenerated SG structures were present in iSGCs-treated mice.
Conclusion
We developed a SG reprogramming strategy to generate functional iSGCs from HDFs by using the single factor EDA in combination with SGM and small molecules. The generation of iSGCs has important implications for future in situ skin regeneration with SG restoration.
... Among all essential mitogens, the insulin/IGF family is the most important factor for hPSC survival and function, and is utilized in various lineage-specific induction protocols, such as neural cell fate induction [14], skin keratinocyte [119] and retinal pigmented epithelium differentiation [9]. IGF signaling also shifts cell fate during mesoderm induction [109]. ...
All living organisms need energy to carry out their essential functions. The importance of energy metabolism is increasingly recognized in human pluripotent stem cells. Energy production is not only essential for cell survival and proliferation, but also critical for pluripotency and cell fate determination. Thus, energy metabolism is an important target in cellular regulation and stem cell applications. In this review, we will discuss key factors that influence energy metabolism and their association with stem cell functions.
... TGFβ signaling mediates mesoderm development, the inhibition of TGFβ signaling is necessary for the fate switch from broblasts or astrocytes to other cells [36,37]. It has been reported that the activation of WNT signaling and BMP signaling and inhibition of TGFβ signaling could elevate CK18, CK14 and CK10 [38]. Interestingly, our results also con rmed the difference of above signaling pathway between broblasts and pSGCs, which suggested targeted regulation of these pathways may enhance the e ciency of sweat gland reprogramming. ...
Background: Large skin defect caused severe disruption to the overall skin structure and irreversible damage of sweat gland (SG), resulting in destroy of physiological function of the skin. Reprogramming fibroblasts into sweat gland lineages may provide a promising strategy to obtain the desirable cell types for functional repair and regeneration of damaged skin.
Methods: A direct reprogramming strategy of single factor ectodermal dysplasia antigen (EDA) in combination with small molecule cocktails promoting cell-fate conversion to regenerate SG cells from human dermal fibroblasts (HDFs) was developed. Quantitative PCR (qPCR), flow cytometry, calcium activity analysis, immunocytochemical analyses and starch-iodine sweat tests were used to characterize the phenotype, gene expression and function features of the induced sweat gland cells (iSGCs).
Results: EDA overexpression drove HDFs toward SG lineages, and HDFs transfected with EDA acquired sweat gland cell phenotype in sweat gland conditional medium (SGM). Small-molecule cocktails favoring SG lineages greatly accelerated the SG fate program in SGM-treated HDF-EDA cells and further induced the regeneration of iSGCs. The HDFs-derived iSGCs exhibited similar phenotypical and functional features of native sweat gland cells. Eventually, in vivo transplantation experiment confirmed that iSGCs had the ability to regenerate SG structurally and functionally.
Conclusion: We developed a SG reprogramming strategy to generate functional iSGCs from HDFs by using single factor EDA in combination with small molecules. The generation of iSGCs has important implications for in situ skin regeneration with restoration of sweat glands in the future.
... The medium itself consists of but is often also supplemented with small molecules and growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), cholera toxin, transforming growth factor beta (TGF-β) inhibitors, ROCK-inhibitor, and insulin in various concentrations throughout the protocol. [104,106,[108][109][110][111][112] These factors are generally used to mimic epidermogenesis but are also included to aid cell proliferation and population doubling. Furthermore, nearly all published protocols also plate cells onto a coating that would mimic the extracellular matrix (ECM), such as native 3D decellularised human dermal FB (HDF) ECM, collagen IV, collagen I or fibronectin. ...
To effectively study the skin and its pathology, various platforms have been used to date, with in vitro 3D skin models being considered the future gold standard. These models have generally been engineered from primary cell lines. However, their short life span leading to the use of various donors, imposes issues with genetic variation. Human pluripotent stem cell (hPSC)‐technology holds great prospects as an alternative to the use of primary cell lines to study the pathophysiology of human skin diseases. This is due to their potential to generate an unlimited number of genetically identical skin models that closely mimic the complexity of in vivo human skin. During the past decade, researchers have therefore started to use human embryonic and induced pluripotent stem cells (hESC/iPSC) to derive skin resident‐like cells and components. These have subsequently been used to engineer hPSC‐derived 3D skin models. In this review, we focus on the advantages, recent developments, and future perspectives in using hPSCs as an alternative cell source for modelling human skin diseases in vitro.
The development of new therapies is hampered by the lack of predictive, and patient-relevant in vitro models. Organ-on-chip (OOC) technologies can potentially recreate physiological features and hold great promise for tissue and disease modeling. However, the non-standardized design of these chips and perfusion control systems has been a barrier to quantitative high-throughput screening (HTS). Here we present a scalable OOC microfluidic platform for applied kinetic in vitro assays (AKITA) that is applicable for high, medium, and low throughput. Its standard 96-well plate and 384-well plate layouts ensure compatibility with existing laboratory workflows and high-throughput data collection and analysis tools. The AKITA plate is optimized for the modeling of vascularized biological barriers, primarily the blood-brain barrier, skin, and lung, with precise flow control on a custom rocker. The integration of trans-epithelial electrical resistance (TEER) sensors allows rapid and repeated monitoring of barrier integrity over long time periods. Together with automated liquid handling and compound permeability testing analyses, we demonstrate the flexibility of the AKITA platform for establishing human-relevant models for preclinical drug and precision medicine's efficacy, toxicity, and permeability under near-physiological conditions.
