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Targeting ectodysplasin promotor by CRISPR/dCas9-effector effectively induces the reprogramming of human bone marrow-derived mesenchymal stem cells into sweat gland-like cells

Background Patients with a deep burn injury are characterized by losing the function of perspiration and being unable to regenerate the sweat glands. Because of their easy accession, multipotency, and lower immunogenicity, bone marrow-derived mesenchymal stem cells (BM-MSCs) represent as an ideal biological source for cell therapy. The aim of this study was to identify whether targeting the promotor of ectodysplasin (EDA) by CRISPR/dCas9-effector (dCas9-E) could induce the BM-MSCs to differentiate into sweat gland-like cells (SGCs). Methods Activation of EDA transcription in BM-MSCs was attained by transfection of naive BM-MSCs with the lenti-CRISPR/dCas9-effector and single-guide RNAs (sgRNAs). The impact of dCas9-E BM-MSCs on the formation of SGCs and repair of burn injury was identified and evaluated both in vitro and in a mouse model. Results After transfection with sgRNA-guided dCas9-E, the BM-MSCs acquired significantly higher transcription and expression of EDA by doxycycline (Dox) induction. Intriguingly, the specific markers (CEA, CK7, CK14, and CK19) of sweat glands were also positive in the transfected BM-MSCs, suggesting that EDA plays a critical role in promoting BM-MSC differentiation into sweat glands. Furthermore, when the dCas9-E BM-MSCs with Dox induction were implanted into a wound in a laboratory animal model, iodine-starch perspiration tests revealed that the treated paws were positive for perspiration, while the paws treated with saline showed a negative manifestation. For the regulatory mechanism, the expression of downstream genes of NF-κB (Shh and cyclin D1) was also enhanced accordingly. Conclusions These results suggest that EDA is a pivotal factor for sweat gland regeneration from BM-MSCs and may also offer a new approach for destroyed sweat glands and extensive deep burns. Electronic supplementary material The online version of this article (doi:10.1186/s13287-017-0758-0) contains supplementary material, which is available to authorized users.


Background
There are millions of patients who suffer the aftereffects of a burn injury every year. Sweat glands are important skin appendages that participate in the regulation of body temperature via an excretory function. Under normal conditions, approximately 25% of heat is eliminated by vaporization of sweat excreted from sweat glands [1]. In the scar tissue of full-thickness burn injury patients, sweat glands cannot undergo a regeneration process. Thus, burn survivors face a long-term problem of secretory function loss and persistent pain. Furthermore, loss of sweat glands leads to the impairment of temperature adjustment which has great influence on life quality for the patients [2]. Therefore, promoting sweat gland transplantation and accelerating the regeneration of sweat glands may be a potential treatment strategy for the patient with a massive deep burn.
Mesenchymal stem cells derived from human bone marrow (BM-MSCs) have been used in stem cell therapy and regeneration medicine to aid wound healing and skin appendage repair [1,3,4]. It has been confirmed that BM-MSCs offer a unique avenue for differentiation into sweat gland-like cells (SGCs) in vitro and can develop secretory function after transplantation in vivo [3,5,6]. However, it takes a long time using conventional induction methods to induce MSCs into SGCs, and the differentiation ratio cannot be guaranteed, which affects their clinical application.
Recently, a RNA-guided precise DNA cleavage technology-Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated system (Cas)-has been widely used in genome editing [7][8][9][10][11], increasing the capacity for stem cell-directed differentiation. Among these, CRISPR/Cas9 provides an accurate and flexible technology for stem cell reprogramming [9]. Most notably, Kearns et al. have developed a doxycycline (Dox)-induced inactive DNA nuclease Cas9-effector (dCas9-E) system to address the differentiation status of stem cells [7]. This dCas9-E system consists of the dCas9 fused to a VP16 tetramer activation domain (VP64) under the control of doxycycline (Dox). Single-guide RNAs (sgRNAs) are then designed to hybridize the target sequence. Finally, these combined elements can generate a DNA complex that recognizes a target locus and activates a specific gene.
Several growth factors have been found to be involved in the development of skin and sweat glands (SGs), such as fibroblast growth factor (FGF) [12], epidermal growth factor (EGF) [13], and anhidrotic ectodysplasias [14,15]. Among these factors, ectodysplasin (EDA) is considered the most important for the development of sweat gland [14,16]. The EDA gene, which belongs to the tumor necrosis factor (TNF) family, has been confirmed to be crucial in skin appendage formation, especially for sweat glands [17]. EDA mainly regulates sweat gland maturation through activating nuclear factor-κB (NF-κB) after combination with the EDA receptor (EDAR) in the early stage of embryonic development [18]. Thus, the EDA gene could be a therapeutic target for MSC reprogramming into SGCs.
In this study, we choose EDA as the key factor which can trigger a cascade reaction in BM-MSC differentiation into sweat gland-like cells in vitro. sgRNA was designed to combine the upstream EDA gene promoter and lentiviral delivery-based systems with a dCas9-E nuclease or with the sgRNAs, and transfected into BM-MSCs. We hypothesized that EDA is a potential factor in BM-MSC differentiation into sweat gland-like cells, and these reprogrammed cells could be applied for the reconstruction of sweat glands and skin wounds.

Animals
Athymic BALB/c nude mice (male, 6-8 weeks old, 20-25 g body weight) were purchased from Vital River (Beijing, China) and housed under pathogen-free conditions. The animal study was performed according to the protocols approved by the Ethics Committee at the General Hospital of the People's Liberation Army and carried out in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines.

Cell culture, chemical reagents and antibodies
Human bone marrow-derived mesenchymal stem cells (BM-MSCs) were purchased from Cyagen Biosciences (HUXMA-90011, Santa Clara, CA, USA) and were cultured in Dulbecco's modified Eagle's Medium (DMEM; Gibco, Grand Island, USA) with low glucose in the presence of 10% fetal bovine serum (FBS; Hyclone, USA). MGC-803 cells were maintained in RPMI 1640 supplemented with 10% FBS. All the cells were incubated in a cell culture incubator at 37°C in a humidified atmosphere containing 5% CO 2 .

Single-guide RNA design and lentiviral vector production
Candidate sgRNAs were identified by searching for 5'-N 20 GG motifs, 276 bases to 26 bases upstream of the EDA transcriptional start site (TSS), that conformed with the nucleotide requirements for the spCas9 PAM recognition element (NGG). The sgRNAs were designed using an online CRISPR Design Tool (http://crispr.mit.edu; Fig. 1c).

Lentiviral production
HEK-293FT cells were maintained in DMEM supplemented with 10% FBS. HEK-293FT cells were split and plated in 10 cm 2 culture dishes. On the following day, packaging plasmids and dCas9-E or sgRNA-coding plasmids were co-transfected using Lipofectamine® 3000 (Invitrogen) according to the manufacturer's instructions.

Generation of stable dCas9-E cell lines and co-expression with sgRNAs
The transduction and selection strategy were as previously described [7]. Briefly, BM-MSCs were incubated with tetracycline-responsive element (TRE)-regulated dCas9-E lentivirus on low-attachment plates. The transduced cells were treated with 1 mg/ml G418 from 48 h after transduction to select and maintain stable cell lines. For experiments utilizing sgRNAs, the stable dCas9-E cell lines were infected with sgRNA lentiviruses. Fortyeight hours later, the sgRNA transduced cells were treated with 1 μg/ml puromycin to select sgRNAexpressing cells and 2 μg/ml doxycycline (Dox) to induce expression of dCas9-E (day 0).

Immunofluorescence
Cells were fixed with 4% paraformaldehyde (30 min) and then incubated in 1% bovine serum albumin (BSA) for 1 h. The cells then incubated with primary anti-human antibodies (1:500) overnight at 4°C. The primary antibodies were decanted and the cells were incubated with phycoerythrin (PE)-conjugated or fluorescein isothiocyanate (FITC)-conjugated goat anti-Rabbit or Mouse IgG secondary antibody (1:1000; Santa Cruz) for 1 h at room temperature in the dark. The cells were finally stained with 4'6-diamidino-2-phenylindole (DAPI; Sigma) and then visualized and examined by a Leica fluorescence microscope.
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) RNA was extracted from BM-MSCs and dCas9-E cells using TRIzol reagent (Invitrogen) at 48 h after Dox (2 μg/ ml) treatment. Total RNA (1.0 μg) was reverse-transcribed into cDNA using ReverTra Ace qRCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan). Each sample was measured in triplicate. The primer sequences of the genes, including CEA, EDA, CK19, Shh, and cyclin D1, are listed in Additional file 1. All genes were normalized to the endogenous reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative expression levels in cells were calculated as fold changes.

Western blot
BM-MSCs and dCas9-E BM-MSCs were harvested after being treated with Dox (2 μg/ml) for 48 h. Cells were lysed in a passive lysis buffer (Promega, Madison, WI, USA) with a cocktail of protease inhibitors (Roche, Mannheim, Germany) at 4°C for 30 min and then centrifuged at 12,000 × g for 10 min at 4°C. Total protein (30 μg) was separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes Fig. 1 Design of CRISPR/dCas9-E nucleases specific for targeting the ectodysplasin (EDA) promotor. a Schematic representation of the EDA genome and b scheme of lentiCRISPR/dCas9-E plasmids. c Schematic representation of the pLKO.1-puro-U6 vector. A guide-sequence insertion site existed downstream of the U6 promoter for cloning the designed single-guide RNA (sgRNA) by the BfuAI restriction site (Millipore, Billerica, MA, USA). After blocking with 5% (w/v) BSA (MP, Auckland, New Zealand) for 1 h, the membranes were probed with primary antibodies overnight at 4°C. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1.5 h. Specific bands were visualized using a luminol reagent (Santa Cruz Biotechnology).

Transmission electron microscopy (TEM)
After Dox treatment, BM-MSCs and dCas9-E BM-MSCs were harvested and fixed with 2.5% glutaraldehyde in phosphate buffer overnight. Cell samples were first dehydrated by a graded series of ethanol for 15 min at each step and transferred to absolute acetone for 20 min. Resin was then used for sample infiltrating and embedding. Samples were prepared as ultrathin sections and observed with the Hitachi TEM system.

Animal studies
BM-MSCs transfected with dCas9-E and pLKO.1-sgRNAs were implanted into a scald injury animal model. The transplantation procedure was performed as previously described [15]. Briefly, full thickness scald injuries were made on both paws of the hind legs of 10 athymic BALB/ c nude mice. The right scalded paw of each mouse received a subcutaneous injection with 1 × 10 6 Dox-induced dCas9-E BM-MSCs in 100 μl medium. The contralateral (sham) scalded paw was subcutaneously injected with saline. The injury sites were photographed and samples collected every week to measure wound healing and re-epithelialization in each group. Twenty days later an iodine-starch perspiration test was performed, and skin biopsies were also examined by histology. Hematoxylin and eosin staining was used for evaluating the extent of re-epithelialization. Masson and Sirius red staining were used to observe paw fibrosis after injury.

Statistical analysis
Statistical analyses were performed using SPSS (V.20) statistical software (SPSS Inc., Chicago, IL, USA). All values are expressed as the mean ± standard deviation. Significant differences were calculated by one-way analysis of variance (ANOVA) followed by the Bonferroni test when performing multiple comparisons between groups. A p value lower than 0.05 was considered as a statistically significant difference.

Design of the EDA-targeting CRISPR/dCas9-E system
The EDA gene, which belongs to the TNF family, has been confirmed to be crucial in sweat gland maturation. Therefore, upregulation of EDA expression may be a feasible way to generate sweat gland cells in vitro. To assess the ability of dCas9-E to upregulate expression of EDA in BM-MSCs, plasmids consisting of a U6 promoter-based lentiviral delivery system for singleguide RNA (sgRNA) to three different target regions upstream of the EDA TSS (Fig. 1a, c) and Dox-inducible expression of dCas9-E under the control of TRE promoters (Fig. 1b) as described by Kearns et al. [7] were obtained from Addgene. An HA marker fused after the dCas9-E protein allowed identification of dCas9-E (Fig. 1b). After identification of the BM-MSCs (Additional file 2), the cells were stable transfected with dCas9-E lentiviral and the HA marker was assessed by immunofluorescence (Fig. 2a) and Western blotting analysis (Fig. 2b).

The transcription and translation of EDA in dCas9-E BM-MSCs
qRT-PCR analysis showed that the levels of EDA gene transcription were significantly increased in dCas9-E BM-MSCs after Dox induction (Fig. 3a). Consistent with the EDA gene expression levels, the Western blot and immunofluorescence results also indicated that EDA protein expression was enhanced by the dCas9-E, and that the sgRNA2 target to the region (-225/-244) of the EDA TSS was more effective than other two sgRNAs (Fig. 3b, c). Thus, the designed sgRNA2 was used in the subsequent experiments.

Dox induced dCas9-E BM-MSCs into sweat gland-like cells in vitro
In order to evaluate the differentiation efficiency of dCas9-E BM-MSCs, cells were analyzed by qRT-PCR and immunofluorescence analysis with the SG biomarkers CEA and CK19, respectively. As expected, dCas9-E BM-MSCs strongly expressed those two SG markers under the induction of Dox; however, BM-MSCs not transfected with dCas9-E were negative (Fig. 4a, c). Furthermore, CEA, CK7, CK14, and CK19 were also detected by Western blotting and those markers were expressed after the induction of Dox (Fig. 4b). TEM examination of cell ultrastructure showed that after Dox induction dCas9-E BM-MSCs had villi structure characteristic of sweat glands (Fig. 4d). These results indicated that dCas9-E BM-MSCs could be induced into SGCs.

Evaluation of dCas9-E BM-MSCs in a sweat gland injured mouse model
A sweat gland injured mouse model was used to examine the effect of dCas9-E BM-MSCs on repair and regeneration of injured tissue. Samples of the injured area were collected every week to evaluate the speed of wound healing. The experimental group treated with Dox-induced dCas9-E BM-MSCs showed a faster healing speed (Fig. 5a), and hematoxylin and eosin staining also demonstrated accelerated re-epithelialization in the experimental group (Additional file 3), with less collagen deposition than in the sham group (Fig. 5b). Immunofluorescence of Ki-67 and CD31 confirmed that dCas9-E BM-MSCs induced by Dox had more proliferation of cells and generated microvascular tissue, resulting in accelerated scarless wound healing (Fig. 5c, d). These results demonstrated that the Dox-induced dCas9-E BM-MSCs can promote wound healing with less fibrosis after injury. At 20 days after treatment with Doxinduced dCas9-E BM-MSCs, eight of ten treated paws were positive for the iodine-starch perspiration test with a distinctive blue and black area. However, the shamtreated paws showed a negative result compared with normal paw skin (Fig. 6a). Hematoxylin and eosin staining showed the duct structure of sweat gland tissue in the experimental and control groups (Fig. 6b). The tissue immunofluorescence from the experimental and control paws indicated that the paw skin treated with Dox-induced dCas9-E BM-MSCs could exhibit the SG biomarkers (Fig. 6c). The number of sweat gland tissues in the experiment group treated with Dox-induced dCas9-E BM-MSCs were significantly higher compared with the sham group (Fig. 6d). These results indicated that Dox-induced dCas9-E BM-MSCs could act as SGCs and promote wound healing.

Tumorigenicity test in animals
The tumorigenicity test for Dox-induced dCas9-E BM-MSCs in mice was conducted at a specific pathogen-free (SPF) facility for 12 weeks. Mice were inspected for tumor development every 3 days for 12 weeks after injection of the indicated cells. No evidence of tumor formation was found in any of the 10 mice treated with Dox-induced dCas9-E BM-MSCs, while 10 out of 10 mice injected with positive control cells (MGC803 cells) grew tumors within 1 month after cell injection (Fig. 7). The results indicated that the induced SGCs did not induce tumors at doses up to 5 × 10 5 cells.

Enhanced expression of Shh and cyclin D1 by activation of EDA
The EDA/EDAR pathway could activate NF-κB through the IKK pathway. Shh and cyclin D1, the downstream genes of NF-κB, were detected by qRT-PCR (Fig. 8a) and Western blotting (Fig. 8b). Since dCas9-E is a Doxinducible protein, the dCas9-E BM-MSCs treated with Dox could express higher levels of Shh and cyclin D1 than those which were not treated with Dox (Fig. 8a, b). Immunofluorescence showed that NF-kB activation increased cyclin D1 and Shh expression in the epidermis and newly generated sweat glands in the experimental group (Fig. 8c). These results indicated that dCas9-E BM-MSCs treated with Dox could activate the NF-κB pathway, enhancing the expression of Shh and cyclin D1.

Discussion
Clinical treatment for, and research into, the structural and functional repair and regeneration of cutaneous tissue after severe burn injury is a huge challenge. Recently, cell therapy has emerged as a new tool to repair the injured skin structure and re-establish the sweating function [1,3,6,15].
With easy accession and culture, a strong differentiation potency, and lower immune resistance, BM-MSCs are considered to be promising cells for cutaneous regeneration in severe burn patients. In the present study, we have demonstrated that direct activation of the EDA gene has great potential for facilitating the formation of sweat gland-like cells via the doxycycline-induced dCas9-E system, giving us a chance to improve the current methods of sweat gland cell generation.
Previous studies have demonstrated that protein encoded by the EDA gene could regulate the development of ectodermal tissues such as sweat glands, hair, and teeth [20]. People who lack the EDA gene have sparse or absent hair and teeth. These results and phenomenon indicate that EDA acts as a key regulator in the initiation stage of cutaneous appendage development [18,21]. Thus, we hypothesized that activation of the EDA gene could facilitate BM-MSC differentiation into sweat glands.
The sgRNA-guided dCas9-E system is a newly developed system to activate transcription of the target gene [7,22]. In the present study, three different sgRNAs were designed to target the -244 bp to -58 bp region upstream of the TSS in the human EDA promoter. Interestingly, individual sgRNAs targeting the EDA promotor around the -244 bp to -58 bp region could effectively increase the transcriptional activation in the presence of dCas9-E (Fig. 3), and the sgRNA2 around the -244 bp to -225 bp region showed higher activity than sgRNA1 (from -131 bp to -112 bp) and sgRNA3 (from -78 bp to -59 bp), suggesting that target position is involved in the activity of the dCas9-E.
Sweat gland epithelial cells have been identified as essential for the construction of skin substitutes and regeneration of sweat glands [23]. Several markers, including CEA, CK7, CK8, CK14, CK15, CK18, and CK19, were identified as the sweat gland-specific markers during SG development. In normal adult skin tissue, these markers (CK7, CK8, CK14, CK15, CK18, and CK19) are expressed in the secretory portion [5,24]. In addition, CEA, a biomarker for colorectal cancer, is also expressed in some normal adult tissue, such as mucous neck cells and pyloric mucous cells in the stomach, and secretory epithelia and duct cells of sweat glands [25]. Therefore, the high expression of CEA, CK7, CK8, CK14, CK15, CK18, and CK19 is considered as an index for the identification of SGs [15,23,26]. After transfection with sgRNA-guided dCas9-E, the BM-MSCs were identified as showing high expression of CEA, CK7, CK14, and CK19 (Fig. 4). These results indicated that activation of the EDA gene could induce BM-MSCs into sweat glandlike cells. The function of the transfected BM-MSCs was measured between treated paws and sham paws in vivo. Results showed that paws treated with Dox-induced dCas9-E BM-MSCs had accelerated wound healing with less collagen deposition (Fig. 5a, b). Primary research has shown that the wound healing process depends on cell proliferation in the basement membrane of the wound edge [27]. Immunofluorescence staining of Ki67 in the Dox-induced dCas9-E BM-MSC treatment group showed higher proliferation in the basement membrane cells compared with the sham group after newly complete re-epithelialization (Fig. 5c), while the number of Ki67-positive cells decreased in the stabilized healing site (Additional file 4), in accordance with changes in MSC-based wound healing therapy [28]. Therefore, we concluded that the transfected cells showed partly preserved MSC features. In addition, hematoxylin and eosin staining and immunofluorescence staining also indicated that the scalded paws treated with Dox-induced dCas9-E BM-MSCs showed sweat gland duct structure and positive markers (CEA and CK19) for sweat glands (Fig. 6). However, the mechanism for EDA regulation of sweat gland development was still unclear.
Previous studies have revealed that multiple signaling pathways, such as ERK-MAPK, EDA/EDAR/NF-κB, and Wnt/β-catenin, were involved in sweat gland development [29,30]. EDA, a member of the TNF superfamily, is one of the functional genes that regulate the development of sweat glands, and mutations in EDA can cause ectodermal dysplasia in humans and lead to sweat-free syndrome [31]. In the present study, therefore, we focused on the EDA/EDAR/NF-κB pathway. The EDA pathway could activate NF-κB through the IKK pathway, and the activated NF-κB can enter the nucleus to promote the expression of cyclin D1, Shh, Fox family genes, and keratins [15], which play an important role in the development of sweat glands [32]. We further found that the EDA/EDAR/NF-κB signaling pathway was activated in the reprogrammed sweat gland-like cells and the injured site in vivo. The activated NF-κB then activates the expression of Shh and cyclin D1 downstream (Fig. 8).
In combination with the in vivo experimental results, we have shown a new therapeutic direction in sweat gland regeneration by revealing a promising technology for direct and effective reprogramming.