In-vitro construction of engineered sweat gland organoids / 解放军医学杂志

Objective To explore a method of constructing engineered sweat gland organoids in vitro by transdifferentiation of epidermal keratinocytes into sweat gland-like cells after lineage re-programming to achieve functional repair of sweat glands. Methods CRISPR/dCas9 system was used to up-regulate the expression of endogenous ectodermal dysplasia ectodysplasin (EDA) genes in human immortalized cuticle keratin HaCaTs, the positive HaCaTs were then screened and cultured. The cells were cultured according to HaCaT group (HaCaT cells cultured in sweat gland medium), HaCaT+Dox group (HaCaT cells cultured in sweat gland medium with 5 μg/ml Dox), HaCaT-E group (HaCaT-E cells cultured in sweat gland medium) and HaCaT-E+Dox group (HaCaT-E cells cultured in sweat gland medium with 5 μg/ml Dox), respectively. RT-PCR and Western blotting were used to detect the expression level of EDA in each group to verify plasmid transfection. In addition, the cells were divided as HaCaT-E group (cultured in sweat gland medium) and HaCaT-E+Dox group (cultured in sweat gland medium with 5 μg/ml Dox) to identify the expression levels of sweat gland-related markers using immunofluorescence staining to verify whether HaCaT were reprogrammed to sweat gland like cells. Furthermore, the sweat gland development microenvironment was imitatively reconstructed using Matrigel as the main extracellular scaffold, which induced sweat gland-like cells to assemble into sweat gland organoids, and the expression of sweat gland surface markers was detected by immunofluorescence staining. After transplantation of sweat gland organoids to mouse scalded paw pads, iodine-starch sweating experiment and histomorphology were performed to detect the involvement of engineered sweat gland organoids in sweat gland regeneration and skin wound repair. Results After transfection with CRISPR/dCas9 lentiviral expression system, positive HaCaT-E cells were obtained. RT-PCR showed the expression level of EDA mRNA in HaCaTE+ Dox group was up-regulated about (4.62±0.19) times than that in HaCaT group (P0.05). Western blotting showed the same trend as did by RT-PCR. After 2-7 days of inducted incubation with sweat gland culture medium containing doxycycline, the cells showed fusiform shape, lumenized networks, as well as up-regulated expression of sweat gland-related markers. After mixedly cultured in a 3-D culture system containing Matrigel for 7 days, the cells proliferated and volume increased to form sweat glands organoids with cystic cavity, and the sweat gland surface markers and functional markers cytokeratin 18 (CK18), a-smooth muscle actin (a-SMA) and aquaporin 5 (AQP5) were positively expressed. In vivo experiments showed that the iodine-starch sweating test was positive in mice, and tissue immunofluorescence staining showed that green fluorescent protein (GFP) positive cells were mainly distributed in the basal layer and subunit basal layer of the epidermis, and the tubular glandular structure was found in the deep and subcutaneous tissues of the dermis, and the GFP and sweat gland cell markers CK18 and a-SMA were observed. Conclusions Up-regulation of endogenous EDA gene by CRISPR/dCas9 system may successfully reprogram HaCaT into sweat gland-like cells. A three-dimensional culture environment constructed with Matrigel as the main extracellular scaffold can induced engineered sweat gland-like cells to self-assembly and develop into sweat gland organoids in vitro. The engineered organoids not only possessed the phenotype and structural characteristics of the sweat gland primordia, but also promoted the regeneration of sweat glands in vivo, thus achieving functional wound repair.

Lineage reprogramming of fibroblasts into induced cardiac progenitor cells by CRISPR/Cas9-based transcriptional activators

Overexpression of exogenous lineage-determining factors succeeds in directly reprogramming fibroblasts to various cell types. Several studies have reported reprogramming of fibroblasts into induced cardiac progenitor cells (iCPCs). CRISPR/Cas9-mediated gene activation is a potential approach for cellular reprogramming due to its high precision and multiplexing capacity. Here we show lineage reprogramming to iCPCs through a dead Cas9 (dCas9)-based transcription activation system. Targeted and robust activation of endogenous cardiac factors, including GATA4, HAND2, MEF2C and TBX5 (G, H, M and T; GHMT), can reprogram human fibroblasts toward iCPCs. The iCPCs show potentials to differentiate into cardiomyocytes, smooth muscle cells and endothelial cells . Addition of MEIS1 to GHMT induces cell cycle arrest in G2/M and facilitates cardiac reprogramming. Lineage reprogramming of human fibroblasts into iCPCs provides a promising cellular resource for disease modeling, drug discovery and individualized cardiac cell therapy.