Transcriptomic and Functional Evidence Show Similarities between Human Amniotic Epithelial Stem Cells and Keratinocytes.

Amniotic epithelial stem cells (AESCs) are considered as potential alternatives to keratinocytes (KCs) in tissue-engineered skin substitutes used for treating skin damage. However, their clinical application is limited since similarities and distinctions between AESCs and KCs remain unclear. Herein, a transcriptomics analysis and functional evaluation were used to understand the commonalities and differences between AESCs and KCs. RNA-sequencing revealed that AESCs are involved in multiple epidermis-associated biological processes shared by KCs and show more similarity to early stage immature KCs than to adult KCs. However, AESCs were observed to be heterogeneous, and some possessed hybrid mesenchymal and epithelial features distinct from KCs. A functional evaluation revealed that AESCs can phagocytose melanosomes transported by melanocytes in both 2D and 3D co-culture systems similar to KCs, which may help reconstitute pigmented skin. The overexpression of TP63 and activation of NOTCH signaling could promote AESC stemness and improve their differentiation features, respectively, bridging the gap between AESCs and KCs. These changes induced the convergence of AESC cell fate with KCs. In future, modified reprogramming strategies, such as the use of small molecules, may facilitate the further modulation human AESCs for use in skin regeneration.

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

BACKGROUND: 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. AIM: To characterize AECs and generate functional hepatic organoids from ASCs and other somatic stem cells. METHODS: AECs, MSCs, and HUVECs were isolated from the placentae and umbilical cords of cesarean section patients. Amnion and primary AEC stemness characteristics and heterogeneity were analyzed by immunocytochemistry, Alkaline phosphatase (AP) staining, and flow cytometry. An adherent AEC subpopulation was selected and evaluated for ASC purification quality by a colony formation assay. AEC transcriptomes were compared with those for other hepatocytes cell sources by bioinformatics. The 2D and 3D culture were compared by relative gene expression using several differentiation protocols. ASCs, MSCs, and HUVECs were combined in a 3D co-culture system to generate hepatic organoids whose structure was compared with a 3D AEC sphere and whose function was elucidated by immunofluorescence imaging, periodic acid Schiff, and an indocyanine green (ICG) test. RESULTS: AECs have certain stemness markers such as EPCAM, SSEA4, and E-cadherin. One AEC subpopulation was also either positive for AP staining or expressed the TRA-1-60 and TRA-1-81 stemness markers. Moreover, it could form colonies and its frequency was enhanced ten-fold in the adherent subpopulation after selective primary passage. Bioinformatics analysis of ribose nucleic acid sequencing revealed that the total AEC gene expression was distant from those of pluripotent stem cells and hepatocytes but some gene expression overlapped among these cells. TJP1, associated with epidermal growth factor receptor, and MET, associated with hepatocyte growth factor receptor, were upregulated and may be important for hepatic differentiation. In conventional flat culture, the cells turned unviable and did not readily differentiate into hepatocytes. In 3D culture, however, hepatic gene expression of the AEC sphere was elevated even under a two-step differentiation protocol. Furthermore, the organoids derived from the MSC and HUVEC co-culture showed 3D structure with polarity, hepatic-like glycogen storage, and ICG absorption/elimination. CONCLUSION: Human amniotic epithelial cells are heterogeneous and certain subpopulations have high stemness. Under a 3D co-culture system, functional hepatic organoids were generated in a multicellular microenvironment.

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

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 MITF+PAX3+TYRP1+ hiMels were localized in the mouse hair bulb and epidermis and produced melanin up to 7 weeks after transplantation, whereas MITF+PAX3+TYRP1- 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.

Ascidiaceibacter salegens gen. nov., sp. nov., isolated from an ascidian.

An aerobic, Gram-stain negative, rod-shaped, non-motile bacterium, designated as strain HQA918T, was isolated from an ascidian, Botryllus schlosseri, which was collected from the coast of Weihai in the north of the Yellow Sea, in China. The strain grew optimally at 28-30 °C, at pH values 7.0-8.0, and in the presence of 1.0-3.0% (w/v) sodium chloride (NaCl). A phylogenetic analysis based on 16S rRNA gene sequences showed that strain HQA918T can be affiliated with the family Flavobacteriaceae in the phylum Bacteroidetes, with 92.7% similarity to its close relatives. The major fatty acids identified were iso-C15:0, iso-C15:0 3-OH, and summed feature 3 (iso-C15:0 2-OH and/or C16:1ω7c). The major polar lipids were phosphatidylethanolamine, one unidentified aminolipid, and five unidentified polar lipids. The G+C content of the genomic DNA was 44.1 mol%. On the basis of the phylogenetic, genotypic, phenotypic, and chemotaxonomic data, this organism should be classified as a representative of a novel genus, for which the name Ascidiaceibacter gen. nov. is proposed. The type species is Ascidiaceibacter salegens sp. nov. (type strain HQA918T = KCTC 52719T = MCCC 1K03259T).