Keratinocyte-like Cells Research Articles

3D Porous Polymer Scaffold-Conjugated KGF-Mimetic Peptide Promotes Functional Skin Regeneration in Chronic Diabetic Wounds.

The re-epithelialization process gets severely dysregulated in chronic nonhealing diabetic foot ulcers/wounds. Keratinocyte growth factor (KGF or FGF-7) is the major modulator of the re-epithelialization process, which regulates the physiological phenotypes of cutaneous keratinocytes. The existing therapeutic strategies of growth factor administration have several limitations. To overcome these, we have designed a KGF-mimetic peptide (KGFp, 13mer) based on the receptor interaction sites in murine KGF. KGFp enhanced migration and transdifferentiation of mouse bone marrow-derived MSCs toward keratinocyte-like cells (KLCs). A significant increase in the expression of skin-specific markers Bnc1 (28.5-fold), Ck5 (14.6-fold), Ck14 (26.1-fold), Ck10 (187.7-fold), and epithelial markers EpCam (23.3-fold) and Cdh1 (64.2-fold) was associated with the activation of ERK1/2 and STAT3 molecular signaling in the KLCs. Further, to enhance the stability of KGFp in the wound microenvironment, it was conjugated to biocompatible 3D porous polymer scaffolds without compromising its active binding sites followed by chemical characterization using Fourier transform infrared spectroscopy, field-emission scanning electron microscopy, dynamic mechanical analysis, and thermogravimetry. In vitro evaluation of the KGFp-conjugated 3D polymer scaffolds revealed its potential for transdifferentiation of MSCs into KLCs. Transplantation of allogeneic MSCGFP using KGFp-conjugated 3D polymer scaffolds in chronic nonhealing type 2 diabetic wounds (db/db transgenic, 50-52 weeks old male mice) significantly enhanced re-epithelialization-mediated wound closure rate (79.3%) as compared to the control groups (Untransplanted -22.4%, MSCGFP-3D polymer scaffold -38.5%). Thus, KGFp-conjugated 3D porous polymer scaffolds drive the fate of the MSCs toward keratinocytes that may serve as potential stem cell delivery platform technology for tissue engineering and transplantation.

Histological and molecular restoration of type VII collagen in Recessive dystrophic epidermolysis bullosa mouse skin by topical injection of keratinocyte-like cells differentiated from human adipose-derived mesenchymal stromal cells

BackgroundRecessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by mutations in the COL7A1 gene, which encodes type VII collagen (COL7), the main constituent of anchoring fibrils for attaching the epidermis to the dermis. Persistent skin erosions frequently result in intractable ulcers in RDEB patients. Adipose-derived mesenchymal stromal cells (AD-MSCs) are easily harvested in large quantities and have low immunogenicity. Therefore, they are suitable for clinical use, including applications involving allogeneic cell transplantation. Keratinocyte-like cells transdifferentiated from AD-MSCs (KC-AD-MSCs) express more COL7 than undifferentiated AD-MSCs and facilitate skin wound healing with less contracture. Therefore, these cells can be used for skin ulcer treatment in RDEB patients. ObjectiveWe investigated whether KC-AD-MSCs transplantation ameliorated the RDEB phenotype severity in the grafted skin of a RDEB mouse model (col7a1-null) on the back of the immunodeficient mouse. MethodsKC-AD-MSCs were intradermally injected into the region surrounding the skin grafts, and this procedure was repeated after 7 days. After a further 7-day interval, the skin grafts were harvested. ResultsNeodeposition of COL7 and generation of anchoring fibrils at the dermal-epidermal junction were observed, although experiments were based on qualitative. ConclusionKC-AD-MSCs may correct the COL7 insufficiency, repair defective/reduced anchoring fibrils, and improve skin integrity in RDEB patients.

Bioengineered MSCCxcr2 transdifferentiated keratinocyte-like cell-derived organoid potentiates skin regeneration through ERK1/2 and STAT3 signaling in diabetic wound

Skin regeneration is severely compromised in diabetic foot ulcers. Allogeneic mesenchymal stem cell (MSC) transplantation is limited due to the poor engraftment, mitogenic, and differentiation potential in the harsh wound microenvironment. Thus, to improve the efficacy of cell therapy, the chemokine receptor Cxcr2 was overexpressed in MSCs (MSCCxcr2). CXCL2/CXCR2 axis induction led to the enhanced proliferation of MSCs through the activation of STAT3 and ERK1/2 signaling. Transcriptional upregulation of FGFR2IIIb (KGF Receptor) promoter by the activated STAT3 and ERK1/2 suggested trans-differentiation of MSCs into keratinocytes. These stable MSCCxcr2 in 2D and 3D (spheroid) cell cultures efficiently transdifferentiated into keratinocyte-like cells (KLCs). An in vivo therapeutic potential of MSCCxcr2 transplantation and its keratinocyte-specific cell fate was observed by accelerated skin tissue regeneration in an excisional splinting wound healing murine model of streptozotocin-induced type 1 diabetes. Finally, 3D skin organoids generated using MSCCxcr2-derived KLCs upon grafting in a relatively avascular and non-healing wounds of type 2 diabetic db/db transgenic old mice resulted in a significant enhancement in the rate of wound closure by increased epithelialization (epidermal layer) and endothelialization (dermal layer). Our findings emphasize the therapeutic role of the CXCL2/CXCR2 axis in inducing trans-differentiation of the MSCs toward KLCs through the activation of ERK1/2 and STAT3 signaling and enhanced skin regeneration potential of 3D organoids grafting in chronic diabetic wounds.

WITHDRAWN: Transdifferentiation of Human Neonatal Foreskin Fibroblasts into Keratinocyte-like Cells via Regulating the AKT/P53/WNT/LEF1 Axis

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Development of a Composite Hydrogel Containing Statistically Optimized PDGF-Loaded Polymeric Nanospheres for Skin Regeneration: In Vitro Evaluation and Stem Cell Differentiation Studies.

Platelet-derived growth factor-BB (PDGF-BB) is a polypeptide growth factor generated by platelet granules faced to cytokines. It plays a role in forming and remodeling various tissue types, including epithelial tissue, through interaction with cell-surface receptors on most mesenchymal origin cells. However, it breaks down quickly in biological fluids, emphasizing the importance of preserving them from biodegradation. To address this challenge, we formulated and evaluated PDGF-encapsulated nanospheres (PD@PCEC) using polycaprolactone-polyethylene glycol-polycaprolactone. PD@PCECs were fabricated through the triple emulsion methodology and optimized by using the Box-Behnken design. The encapsulation efficiency (EE) of nanoencapsulated PDGF-BB was investigated concerning four variables: stirring rate (X1), stirring duration (X2), poly(vinyl alcohol) concentration (X3), and PDGF-BB concentration (X4). The selected optimized nanospheres were integrated into a gelatin-collagen scaffold (PD@PCEC@GC) and assessed for morphology, biocompatibility, in vitro release, and differentiation-inducing activity in human adipose-derived stem cells (hADSCs). The optimized PD@PCEC nanospheres exhibited a particle size of 177.9 ± 91 nm, a zeta potential of 5.2 mV, and an EE of 87.7 ± 0.44%. The release profile demonstrated approximately 85% of loaded PDGF-BB released during the first 360 h, with a sustained release over the entire 504 h period, maintaining bioactivity of 87.3%. The study also included an evaluation of the physicochemical properties of the scaffolds and an assessment of hADSC adhesion to the scaffold's surface. Additionally, hADSCs cultivated within the scaffold effectively differentiated into keratinocyte-like cells (KLCs) over 21 days, evidenced by morphological changes and upregulation of keratinocyte-specific genes, including cytokeratin 18, cytokeratin 19, and involucrin, at both transcriptional and protein levels.

Human-induced pluripotent stem cell-derived keratinocyte-like cells for research on protease-activated receptor 2 in non-histaminergic cascades of atopic dermatitis.

Keratinocytes are the most abundant cells in the epidermis, and as part of the frontline immunological defense system, keratinocytes function as a barrier to exogenous attacks. Protease-activated receptor 2 (PAR2) is expressed in human keratinocytes and activated in several inflammatory conditions, such as atopic dermatitis (AD). In this study, we demonstrated the differentiation of human induced pluripotent stem cell (iPSC) into keratinocytes by the improved robust procedure, and confirmed that human-induced pluripotent stem cell-derived keratinocyte-like cells (iKera) express PAR2, which is activated by external addition of the ligand peptide and trypsin. The activation of PAR2 led to the release of calcium from intracellular calcium storage, followed by the release of the pro-inflammatory cytokine, tumor necrosis factor-alpha. Moreover, PAR2 antagonist, I-191, inhibited calcium release in a dose-dependent manner. This is the first study to demonstrate that iKera expresses a functional PAR2 protein. Furthermore, our results indicate cross-talk between the PAR2- and IL-4-mediated inflammatory axes in iKera, suggesting that iKera can be used as a platform for a broad range of mechanism-targeted drug screening in AD. Significance Statement This is the first study to provide evidence that human-induced pluripotent stem cell-derived keratinocyte-like cells (iKera) express functional protease-activated receptor 2 (PAR2). Furthermore, we demonstrated in iKera that the IL-4 inflammatory axis can cross-talk with the PAR2 mediated inflammatory axis in keratinocytes. To the best of our knowledge, this is the first report to indicate that iKera can be used for research and as a drug screening platform for atopic dermatitis (AD).

Direct chemical induction of hepatocyte-like cells with capacity for liver repopulation.

Cell fate can be directly reprogrammed from accessible cell types (e.g., fibroblasts) into functional cell types by exposure to small molecule stimuli. However, no chemical reprogramming method has been reported to date that successfully generates functional hepatocyte-like cells that can repopulate liver tissue, casting doubt over the feasibility of chemical reprogramming approaches to obtain desirable cell types for therapeutic applications. Here, through chemical induction of phenotypic plasticity, we provide a proof-of-concept demonstration of the direct chemical reprogramming of mouse fibroblasts into functional hepatocyte-like cells using exposure to small molecule cocktails in culture medium to successively stimulate endogenous expression of master transcription factors associated with hepatocyte development, such as hepatocyte nuclear factor 4a, nuclear receptor subfamily 1, group I, member 2, and nuclear receptor subfamily 1, group H, member 4. RNA sequencing analysis, metabolic assays, and in vivo physiological experiments show that chemically induced hepatocytes (CiHeps) exhibit comparable activity and function to primary hepatocytes, especially in liver repopulation to rescue liver failure in fumarylacetoacetate hydrolase -/- recombination activating gene 2 -/- interleukin 2 receptor, gamma chain -/- mice in vivo . Single-cell RNA-seq further revealed that gastrointestinal-like and keratinocyte-like cells were induced along with CiHeps, resembling the activation of an intestinal program within hepatic reprogramming as described in transgenic approaches. Our findings show that direct chemical reprogramming can generate hepatocyte-like cells with high-quality physiological properties, providing a paradigm for establishing hepatocyte identity in fibroblasts and demonstrating the potential for chemical reprogramming in organ/tissue repair and regeneration therapies.

Electrospun gelatin/methylcellulose hybrid nanofibers promoted the maturation of human cutaneous tissue progenitor cells toward keratinocyte-like cells

Electrospun gelatin/methylcellulose hybrid nanofibers promoted the maturation of human cutaneous tissue progenitor cells toward keratinocyte-like cells

Generation of a lung squamous cell carcinoma three-dimensional culture model with keratinizing structures

Tumor nests in lung squamous cell carcinoma (LUSC) have a hierarchical structure resembling squamous epithelium. The nests consist of basal-like cells on the periphery and layers of keratinocyte-like cells that differentiate towards the center of the nest, forming keratin pearls. Reproducing this spatial heterogeneity in in vitro models would be useful for understanding the biology of LUSC. Here, we established a three-dimensional (3D) culture model with a squamous epithelial structure using LUSC cell lines PLR327F-LD41 and MCC001F, established in-house. When PLR327F-LD41 cells were cultured in a mixture of Matrigel and collagen I, they generated 3D colonies (designated cancer organoids, or COs) with involucrin (IVL)-positive keratinizing cells in the center (IVLinner COs). COs with uniform size were generated by seeding PLR327F-LD41 cells in a form of small cell aggregates. Since Notch signaling induces the differentiation of squamous epithelium, we confirmed the effect of γ-secretase inhibitor in inhibiting Notch signaling in IVLinner COs. Surprisingly, γ-secretase inhibitor did not block induction of IVL-positive cells; however, cells residing between the CK5-positive basal-like layer and IVL-positive layer decreased significantly. Thus, our 3D culture model with uniform size and structure promises to be a useful tool for elucidating the biology of LUSC and for screening drug-candidates.

Preparation, characterization, and antibacterial properties of hybrid nanofibrous scaffolds for cutaneous tissue engineering

Since polymeric nanofibrous scaffolds have been widely used in tissue regeneration, the risk of bacterial infections should not be neglected. In the present work, poly-caprolactone-silk fibroin-soluble eggshell membrane-silver nanoparticles (PCL-SF-SESM-AgNPs) and caprolactone-silk fibroin-soluble eggshell membrane-chitosan (PCL-SF-SESM-CS) scaffolds were fabricated via the electrospinning method for cutaneous regeneration. The composition, morphology, hydrophilicity, and mechanical features of prepared scaffolds were evaluated using Fourier transform infrared (FT-IR), scanning electron microscope (SEM), tensile, and water contact angle tests. The existence of AgNPs in PCL/SF/SESM/AgNPs nanofibers was confirmed by UV-visible, Transmission electron microscopes (TEM), and X-Ray Diffraction (XRD) patterns. Besides, cell adhesion, proliferation, and differentiation process of cutaneous progenitor cells, namely basal cell carcinoma (BCCs), toward keratinocyte-like cells were evaluated using MTT analysis, DAPI, Immunofluorescence imaging (IF), and Real-Time Quantitative Reverse Transcription PCR (QRT-PCR) assay. The results indicated that prepared nanofibrous mats are appropriate candidates for cutaneous regeneration and in advanced in vivo applications could be used. Lastly, the antimicrobial potential of prepared nanofibers against microorganisms such as E. coli, S. aureus, and C. Albicans was analyzed using the disc diffusion method. Results revealed that chitosan-containing nanofibrous scaffolds indicate inhibition against S. aureus, but PCL-SF-SESM as control group not. In addition, against C. albicans any antifungal activity was not observed.

HiPSC-Derived Epidermal Keratinocytes from Ichthyosis Patients Show Altered Expression of Cornification Markers.

Inherited ichthyoses represent a large heterogeneous group of skin disorders characterised by impaired epidermal barrier function and disturbed cornification. Current knowledge about disease mechanisms has been uncovered mainly through the use of mouse models or human skin organotypic models. However, most mouse lines suffer from severe epidermal barrier defects causing neonatal death and human keratinocytes have very limited proliferation ability in vitro. Therefore, the development of disease models based on patient derived human induced pluripotent stem cells (hiPSCs) is highly relevant. For this purpose, we have generated hiPSCs from patients with congenital ichthyosis, either non-syndromic autosomal recessive congenital ichthyosis (ARCI) or the ichthyosis syndrome trichothiodystrophy (TTD). hiPSCs were successfully differentiated into basal keratinocyte-like cells (hiPSC-bKs), with high expression of epidermal keratins. In the presence of higher calcium concentrations, terminal differentiation of hiPSC-bKs was induced and markers KRT1 and IVL expressed. TTD1 hiPSC-bKs showed reduced expression of FLG, SPRR2B and lipoxygenase genes. ARCI hiPSC-bKs showed more severe defects, with downregulation of several cornification genes. The application of hiPSC technology to TTD1 and ARCI demonstrates the successful generation of in vitro models mimicking the disease phenotypes, proving a valuable system both for further molecular investigations and drug development for ichthyosis patients.

Formalin-killed Propionibacterium acnes activates the aryl hydrocarbon receptor and modifies differentiation of SZ95 sebocytes in vitro

Acne vulgaris is a common pilosebaceous disease associated with Propionibacterium acnes (P. acnes). Resolution of comedones may occur in association with shrunken sebaceous glands (SGs) containing de-differentiated cells, however the role of P. acnes is unclear.To investigate the effects of P. acnes on aryl hydrocarbon receptor (AhR) activation, lipogenesis and differentiation in cultured immortalized human SZ95 sebocytes.Cultured sebocytes were incubated with formalin-killed (f-) P. acnes (f-P. acnes) at different ratios of multiplicity of infection. The mRNA levels of the AhR downstream cytochrome P450 (CYP) genes were measured by quantitative RT-PCR, nuclear translocation of AhR by western blot and immunofluorescence, lipogenesis and keratinization by gene set enrichment analysis (GSEA), lipid related analysis by Oil red O staining and Nile red staining, and sebaceous differentiation-related gene expression by western blot.f-P. acnes upregulated CYPs mRNA levels and induced translocation of AhR protein from the cytoplasm into the nucleus. GSEA revealed downregulation of lipogenesis and upregulation of keratinization. f-P. acnes inhibited linoleic acid-induced neutral lipid synthesis and expression of sebocyte markers, keratin 7 and mucin1/EMA, but increased expression of keratinocyte markers, keratin 10 and involucrin, which were abolished by AhR gene silencing. Inhibition of lipogenesis-related genes, such as sterol response element-binding protein, was also observed.f-P. acnes inhibits lipogenesis and induces terminal differentiation of sebocytes, into keratinocyte-like cells, via activation of the AhR pathway in vitro, suggesting that follicular P. acnes is not only acnegenic but also promotes acne remission through feedback regulation of sebum production.

Keratinocyte-Like Cells Trans-Differentiated from Human Adipose-Derived Stem Cells, Facilitate Skin Wound Healing in Mice.

BackgroundMesenchymal stem cells (MSCs) have been reported to promote wound healing in both animal models and human studies. Among MSCs, adipose-derived stem cells (ADSCs) can be easily harvested in large quantities.ObjectiveWe investigated whether skin wound healing in mice can be facilitated by keratinocyte-like cells differentiated from ADSCs (KC-ADSCs).MethodsFor the wound contraction and epithelialization model, a 20 mm×20 mm fullthickness skin wound was made on the dorsum. For the wound epithelialization model, a 6 mm×6 mm full-thickness skin wound was made on the dorsum. A nitrile rubber stent with an inner diameter of 8 mm was sutured around the wounds to minimize wound contraction. Undifferentiated ADSCs (uADSCs) or KC-ADSCs was injected around the wound base in both models. To evaluate whether the injected ADSCs could enhance wound contraction in a skin wound, the contractile activity of ADSCs was assessed by an in vitro type I collagen gel contraction assay. Alpha-smooth muscle actin (αSMA) expressions in uADSCs and KC-ADSCs were also evaluated by flow cytometry and real-time polymerase chain reaction.ResultsIn a wound contraction and epithelialization model, KC-ADSCs further facilitated wound healing compared with uADSCs. In a wound epithelialization model, KC-ADSCs also further facilitated wound epithelialization compared with uADSCs. The contractile activity of KC-ADSCs was lower than that of uADSCs. The uADSCs expressed high levels of αSMA, which decreased after the differentiation into keratinocyte-like cells.ConclusionOur results suggest that the wound healing effect of KC-ADSCs depends primarily on re-epithelialization rather than wound contraction.

Cellular heterogeneity landscape in laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is a highly malignant tumor originated from respiratory system. Although there have been many improvements in therapy until now, reducing the high mortality remains difficult. Understanding the cellular heterogeneity of LSCC could contribute to improve this problem. Single-cell RNA sequencing was applied to dissect the cell composition and molecular characteristics of LSCC tissues. Immunohistochemistry staining of the LSCC tissues was performed to identify the spatial location of tumor cells. Survival analysis of marker genes was executed in The Cancer Genome Atlas to verify the correlation between each cell clusters and patients' prognosis. The LSCC tissue cells were finely grouped into various clusters, including tumor cells, immune cells, epithelial cells, fibroblasts and endothelial cells. Notably, in tumor cells, keratinocyte-like cells were in the core of tumor while malignant proliferating cells were located at the tumor edge. The malignant proliferating cells were correlated with poor prognosis. In summary, this is the first study to delineate a landscape of the LSCC intratumor heterogeneity. Our work might help researchers have a better understanding for tumor progression.

Long-term expansion of directly reprogrammed keratinocyte-like cells and in vitro reconstitution of human skin

BackgroundHuman keratinocytes and derived products are crucial for skin repair and regeneration. Despite substantial advances in engineered skin equivalents, their poor availability and immunorejection remain major challenges in skin grafting.MethodsInduced keratinocyte-like cells (iKCs) were directly reprogrammed from human urine cells by retroviral transduction of two lineage-specific transcription factors BMI1 and △NP63α (BN). Expression of keratinocyte stem cell or their differentiation markers were assessed by PCR, immunofluorescence and RNA-Sequencing. Regeneration capacity of iKCs were assessed by reconstitution of a human skin equivalent under air-interface condition.ResultsBN-driven iKCs were similar to primary keratinocytes (pKCs) in terms of their morphology, protein expression, differentiation potential, and global gene expression. Moreover, BN-iKCs self-assembled to form stratified skin equivalents in vitro.ConclusionsThis study demonstrated an approach to generate human iKCs that could be directly reprogrammed from human somatic cells and extensively expanded in serum- and feeder cell-free systems, which will facilitate their broad applicability in an efficient and patient-specific manner.

Epithelial differentiation of human adipose-derived stem cells (hASCs) undergoing three-dimensional (3D) cultivation with collagen sponge scaffold (CSS) via an indirect co-culture strategy

BackgroundThree-dimensional (3D) cultivation with biomaterials was proposed to facilitate stem cell epithelial differentiation for wound healing. However, whether human adipose-derived stem cells (hASCs) on collagen sponge scaffold (CSS) better differentiate to keratinocytes remains unclear.Methods3D cultivation with CSS on hASC epidermal differentiation co-cultured with HaCaT cells at air-liquid interface (ALI) was compared with two-dimensional (2D) form and cultivation without “co-culture” or “ALI.” Cellular morphology, cell adhesion, and growth condition were evaluated, followed by the protein and gene expression of keratin 14 (K14, keratinocyte specific marker).ResultsTypical cobblestone morphology of keratinocytes was remarkably observed in co-cultured hASCs at ALI, but those seeded on the CSS exhibited more keratinocyte-like cells under an invert microscope and scanning electron microscope. Desired cell adhesion and proliferation were confirmed in 3D differentiation groups by rhodamine-labeled phalloidin staining, consistent with H&E staining. Compared with those cultured in 2D culture system or without “ALI,” immunofluorescence staining and gene expression analysis revealed hASCs co-cultured over CSS expressed K14 at higher levels at day 15.ConclusionsCSS is positive to promote epithelial differentiation of hASCs, which will foster a deeper understanding of artificial dermis in skin wound healing and regeneration.

Skeletal meroterpenoids from Ganoderma petchii mushrooms that potentially stimulate umbilical cord mesenchymal stem cells.

(±)-Petchilactones A-C (1-3), three pairs of enantiomeric meroterpenoids respectively with a 6/6/5/5 or a 5/5/5/7/6 ring system were isolated from Ganoderma petchii. Their structures including absolute configurations were assigned by using spectroscopic, computational, and X-ray diffraction methods. Compounds 1 and 2 represent a new skeletal meroterpenoid. Biological evaluation found that (-)-1 and (-)-3 could induce umbilical cord mesenchymal stem cells into keratinocyte-like cells.

A novel egg-shell membrane based hybrid nanofibrous scaffold for cutaneous tissue engineering

BackgroundThe main issue in cutaneous regeneration is to develop engineered scaffolds based on natural extracellular matrix to promote dynamics of skin progenitor cells and accelerate differentiation into mature keratinocytes.MethodsIn this study, nanofibrous scaffolds composed of a blend poly (ɛ-caprolactone) (PCL), silk fibroin (SF), soluble eggshell membrane (SESM), and Aloe vera (AV) gel were developed by electrospinning method and human basal cells were used to examine differentiation capacity toward keratinocyte-like cells. For this propose, cells were allocated to four distinct groups; control, PCL/SF, PCL/SF/SESM, and PCL/SF/SESM/AV. In all groups, cells were incubated with differentiation medium. Morphology, composition, hydrophilicity and mechanical features of PCL/SF, PCL/SF/SESM and PCL/SF/SESM/AV nanofibers were studied by scanning electron microscopy (SEM), Fourier transforms infrared spectroscopy (FT-IR), water contact angle and tensile tests. To examine the orientation of basal cells to mature keratinocytes, we performed immunofluorescence analysis by monitoring cytokeratin-19. The expression of genes such as involucrin, keratin-14 and -5 was monitored by real-time PCR assay.ResultsPCL/SF, PCL/SF/SESM, and PCL/SF/SESM/AV had suitable physic chemical indices and biological activities to be applied as biomimetic scaffolds for the restoration cutaneous tissue. Compared to control, we found an increased basal cell proliferation at 7 and 14 days after plating on scaffolds and reach maximum levels in group PCL/SF/SESM/AV on day 14 (p < 0.05). Electron microscopy showed cell flattening, morphological adaptation. An integrated cell-to-cell connection was generated after cell seeding on scaffolds in all groups. Immunofluorescence imaging showed the ability of basal cells to synthesize cytokeratin-19 in PCL/SF, PCL/SF/SESM, and positive control cells after exposure to differentiation medium. However, these values were less in PCL/SF/SESM/AV compared to other groups. Real-time PCR analysis showed the potency of all scaffolds to induce the transcription of involucrin, keratin-14 and -5, especially involucrin in PCL/SF/SESM/AV group compared to the negative control.ConclusionModulation of scaffolds with natural biopolymers could enable us to synthesize structures appropriate for cutaneous regeneration.

Bioengineering of Fetal Skin: Differentiation of Amniotic Fluid Stem Cells into Keratinocytes

Purpose: Open fetal spina bifida repair has become a novel clinical standard of care. In very large spina bifida lesions, the skin defect cannot be covered primarily, asking for alternative solutions. We hypothesize that amniotic fluid stem cells (AFSC) could be differentiated into keratinocytes that could then be used to bioengineer autologous skin usable for in utero back coverage. Methods: To obtain human AFSC, amniotic fluid samples obtained from fetal surgeries were subjected to immunoselection for c-kit. C-kit-positive samples and controls were cultured with the additives morphogenetic protein 4 and vitamin C to induce differentiation towards keratinocytes. This process was monitored by measuring the expression of K8 and K14 via immunohistochemical staining, flow cytometry, and polymerase chain reaction. Results: After immunoselection and expansion, most cells were positive for K8, but none for K14. After completion of the differentiation protocol, cell colonies with keratinocyte-like appearance could be observed, but cells remained positive for K8 and negative for K14, indicating failed differentiation into keratinocytes. Conclusions: Culturing of keratinocyte-like cells from AFSC, harvested intraoperatively, was not feasible in this setting. The reasons for failure must be investigated and eliminated, as bioengineering of fetal skin for clinical use during fetal surgery for spina bifida remains an attractive goal.

Spontaneous evolution of human skin fibroblasts into wound-healing keratinocyte-like cells.

Producing keratinocyte cells (KCs) in large scale is difficult due to their slow proliferation, disabling their use as seed cells for skin regeneration and wound healing. Cell reprogramming is a promising inducer-based approach to KC production but only reaches very low cellular conversion. Here we reported a unique cellular conversion phenomenon, where human skin fibroblasts (FBs) were spontaneously converted into keratinocyte-like cells (KLCs) over the time without using any inducers.Methods: FBs were routinely cultured for more than 120 days in regular culture medium. Characteristics of KLCs were checked at the molecular and cellular level. Then the functionality and safety of the KLCs were verified by wound healing and tumorigenicity assay, respectively. To identify the mechanism of the cell conversion phenomenon, high-throughput RNA sequencing was also performed.Results: The global conversion started on day 90 and reached 90% on day 110. The KLCs were as functional and effective as KCs in wound healing without causing oncogenicity. The conversion was regulated via a PI3K-AKT signaling pathway mediated by a long non-coding RNA, LINC00672. Modulating the pathway could shorten the conversion time to 14 days.Conclusion: The discovered FBs-KLCs conversion in the study might open a new avenue to the scalable production of cell sources needed for regenerating skins and healing large-area wounds.