1232 Diabetes Volume 69, June 2020

TWIST1-Reprogrammed Endothelial Cell Transplantation Potentiates Neovascularization-Mediated Diabetic Wound Tissue Regeneration

Komal Kaushik and Amitava Das

Diabetes 2020;69:1232–1247 | https://doi.org/10.2337/db20-0138

Hypovascularized diabetic nonhealing wounds are due to Endothelial progenitor cells (EPC) are the key cellular reduced number and impaired physiology of endogenous effectors that have the potential to differentiate into endothelial progenitor cell (EPC) population that limits their endothelial cells (EC) during postnatal neovasculariza- recruitment and mobilization at the wound site. For enrich- tion (1). Neovascularization is often compromised due ment of the EPC repertoire from nonendothelial precursors, to reduced number of EPC in diabetic conditions thereby abundantly available mesenchymal stromal cells (MSC) limiting the endogenous enrichment or autologous EPC were reprogrammed into induced endothelial cells (iEC). transplantation therapies (2–4). De novo EC generation fi We identi ed cell signaling molecular targets by meta- from nonendothelial precursor cells could be a promising analysis of microarray data sets. BMP-2 induction leads strategy to improve neovascularization to increase the – to the expression of inhibitory Smad 6/7 dependent neg- EPC repertoire. Studies have been attempted to modulate ’ ative transcriptional regulation of ID1, rendering the latter s the fate of induced pluripotent stem cells (iPSC) or embry- reduced binding to TWIST1 during transdifferentiation of onic stem cells toward EC by differentiation using specific Wharton jelly–derived MSC (WJ-MSC) into iEC. TWIST1, in growth factors (5) or direct reprogramming of fibroblast/ turn, regulates endothelial gene transcription, positively of somatic cells toward endothelial lineage by overexpressing proangiogenic KDR and negatively, in part, of antian- endothelial-specific transcription factors (6–8). Han et al. giogenic SFRP4. Twist1 reprogramming enhanced the (9) depicted conversion of only 4% of murine skin fibro- endothelial lineage commitment of WJ-MSC and in- blasts into EC by forced expression of defined EC-specific creased the vasculogenic potential of reprogrammed en- ’ dothelial cells (rEC). Transplantation of stable TWIST1 rEC transcription factors. These approaches of EC sgeneration IMMUNOLOGY AND TRANSPLANTATION into a type 1 and 2 diabetic full-thickness splinted wound are limited by their low reprogramming/transdifferentia- fi – healing murine model enhanced the microcirculatory blood tion ef ciency. Similarly, multiple E26 transformation fi flow and accelerated the wound tissue regeneration. An speci c (ETS)-related transcription factors were used to increased or decreased colocalization of GFP with KDR/ reprogram somatic cells into EC, which are known potent SFRP4 and CD31 in the regenerated diabetic wound bed regulators of vascular development and angiogenesis. How- with TWIST1 overexpression or silencing (piLenti-TWIST1- ever, stable proliferative EC were not obtained because of shRNA-GFP), respectively, further confirmed improved the lack of precise temporal control on gene overexpression neovascularization. This study depicted the reprogram- (10,11). On the contrary, another independent study reported ming of WJ-MSC into rEC using unique transcription that overexpression of a single ETS-related transcription factor TWIST1 for an efficacious cell transplantation factor, ETV2, was sufficient to reprogram human fibro- therapy to induce neovascularization-mediated dia- blasts into EC (12,13). However, it was concluded that too betic wound tissue regeneration. low or high levels of ETV2 expression led to compromised

Department of Applied Biology, Council of Scientific and Industrial Research– This article contains supplementary material online at https://doi.org/10.2337/db20-4567/ Indian Institute of Chemical Technology, Hyderabad, India suppl.12030417. fi fi Academy of Scienti c and Innovative Research, Council of Scienti c and Industrial © 2020 by the American Diabetes Association. Readers may use this article as – Research Indian Institute of Chemical Technology Campus, Hyderabad, India long as the work is properly cited, the use is educational and not for profit, and the Corresponding author: Amitava Das, [email protected] work is not altered. More information is available at https://www.diabetesjournals Received 6 February 2020 and accepted 23 March 2020 .org/content/license. diabetes.diabetesjournals.org Kaushik and Das 1233 endothelial reprogramming. Therefore, specificoptimi- various statistical packages from the Bioconductor suite zation of combination or expression levels of target (https://www.bioconductor.org). Principal component anal- cell–specific transcription factors is required for success- ysis was performed using Partek Genomics Suite (https://www ful endothelial reprogramming of cells. Nonetheless, .partek.com) to identify the close relationship between the Takahashi et al. (14) also revealed that reprogramming sample groups used in the study. The differentially efficiency of iPSC from somatic cells was even lower i.e., expressed genes (DEGs) were determined between each ;0.1%, thereby necessitating the use of other stem cell group by the limma package (Linear Model for Microarray types for EC generation. Interestingly, adult stem cells such Analysis). Next, an empirical Bayesian approach was used to as mesenchymal stem/stromal cells (MSC) have been sug- calculate t-statistic for each group followed by the use of gested to be a reliable source for induced generation of Benjamini-Hochberg algorithm for multiple testing error to various other cell types apart from its trilineage—chondrocyte, calculate the corrected P values. Selection of up- and down- adipocyte, or osteoblast—due to its unique characteristic regulated genes with log fold change by 2 were sorted from of plasticity; i.e., they can be transdifferentiated into each group and subjected to annotation using the “anno- nonobvious lineages using molecular or pharmacological tate” package. To find the pathways associated with the top approaches. However, transdifferentiation of MSC into DEGs, we imported the up- and downregulated genes from induced EC (iEC) has been explored using EC-specific each array in Protein ANalysis THrough Evolutionary Re- growth factors only till date (15–17). Also, MSC are lationship (PANTHER) software to classify them based on the most abundantly available adult stem cell types their participation in signaling pathways relevant to vascu- that can be harvested for various types of human tissues: larization (19). Heat maps were generated using expression bone marrow, adipose, umbilical cord, blood, etc. Thus, values of upregulated and downregulated genes with the help the reprogramming of MSC into induced EC (iEC) using of Gitools software to identify genes showing increased molecular tools could be a promising strategy for the or decreased expression profile during the transdifferen- generation of patient-specific EC for autologous trans- tiation of MSC to EPC and EPC to mature EC. Furthermore, plantation therapy. Recently, we demonstrated that in- self-organizing map clustering was performed using Partek hibition of cyclo-oxygenase 2 (Cox-2) enzyme using Genomics Suite software to identify the gene clusters with specific inhibitor potentiated transdifferentiation a consistent increase or decrease in gene expression profile of Wharton jelly–derived MSC (WJ-MSC) into EC (20). Protein-protein interaction network was also gener- in vitro, and its transplantation demonstrated an improved ated using STRING v9.1 software (https://string-db.org) to vascularization-mediated translational efficiency in vivo determine the existing information about the interactions (18). The current study used the meta-analyses of micro- between selected genes (21,22) for further in vitro analysis. array data sets along with molecular and cellular tools for Cell Culture Studies gaining insights into the transcriptional regulation oc- A well-characterized human WJ-MSC (HiMedia, Mumbai, curring during transdifferentiation of WJ-MSC into iEC followed by developing a novel strategy for generating India) was cultured to perform experiments within pas- sages five to seven in MSC expansion medium (HiMedia) as reprogrammed EC (rEC) by forced overexpression of a single previously described (18). transcription factor in WJ-MSC. Furthermore, in vivo mu- rine diabetic nonhealing wound models were used to eval- Transdifferentiation of WJ-MSC Into iEC fi uate the translational ef ciency of these rEC to enhance WJ-MSC were subjected for transdifferentiation into EC neovascularization during skin tissue regeneration. by culturing in endothelial growth medium (EBM-2; Lonza) for 7 days (iEC-D7) and 14 days (iEC-D14) as previously RESEARCH DESIGN AND METHODS described (18). Morphological characterization of WJ-MSC Meta-analysis of Microarray Data Sets and iEC was performed using bright-field microscopy fi The raw data les of mRNA expression data sets for three at 310 magnification (Olympus). Separately, the trans- microarray studies available in the National Center for differentiation of WJ-MSC into iEC-D7 was also per- Biotechnology Information Gene Expression Omnibus formed in the absence/presence of BMP2 (10 ng/mL; (NCBI-GEO) (https://www.ncbi.nlm.nih.gov/geo) and ArrayEx- HiMedia) along with BMP2 receptor inhibitor, DMH1 press (https://www.ebi.ac.uk/arrayexpress/) were retrieved to (100 nmol/L; (HiMedia) (15–17). compare the gene expression profile in different human cell types such as 1) MSC (E-MEXP-3071), 2) EPC (GSE37045 and DiI Ac-LDL Uptake Assay GSE12891), and 3) human umbilical vein EC (HUVEC) The WJ-MSC and iEC-D7 and -14 were incubated with DiI (GSE25979) using Affymetrix Hugene-1.0.stv1, Affymetrix Ac-LDL (1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocya- hgu133plus2, and Affymetrix HuEx-1-0-st, respectively. nine acetylated LDL) (5 mg/mL) containing respective me- Since these microarrays were performed using different plat- dium for evaluation of its rate of uptake as described forms consisting of different numbers of genes, we performed previously (18). In another set of experiments, the effect an initial data quality check, preprocessing, and normal- of BMP2 during transdifferentiation of WJ-MSC into iEC ization of each data set separately using the R program- was evaluated by comparing the level of DiI Ac-LDL uptake ming language (https://www.r-project.org) installed with in these cells. 1234 Reprogramming in MSC Drives Fate Toward rEC Diabetes Volume 69, June 2020

Gene Expression Studies (abm) and/or control vector, piLenti-Scr-siRNA-GFP (abm). For evaluation of expression levels of endothelial-specific 24 h posttransfection, the medium was changed into either gene markers in WJ-MSC and iEC-D7 and -D14, quanti- MSC expansion medium or EGM-2 medium as described tative real-time PCR (qPCR) was performed as previously above. Transfection efficiency was monitored by evaluating described (18) using gene-specificprimersofKDR, VEGFR1, the GFP expression followed by confirmation of transgene CD31, VE-Cadherin, CD146, VWF, ANGPT2, TIE-2, EDN1, overexpression and/or silencing using Western blot analysis EDNRB, NRP1, NRP2, eNOS,andCD105. The gene expres- for TWIST1, FLAG, or GFP as previously described (24). sion values were normalized to the housekeeping gene, eukaryotic 18S rRNA, and values represented as fold change Promoter-Reporter Dual Luciferase Activity Assay 4 relative to WJ-MSC as control. WJ-MSC at a cell density of 10 cells/well were plated in a 96-well plate and incubated for 24 h at 37°C. Cells were Immunofluorescence Studies transfected with pMCS-Green Renilla Luc vector subcloned Cultured WJ-MSC and iEC-D7 and -D14 were washed, with the desired promoter region and internal control vector fixed, and permeabilized before separate incubation with cytomegalovirus (CMV)-Red Firefly Luc control plasmid primary antibodies against KDR, eNOS, and/or CD31 fol- (pCMV-Red FireflyLuc) using Lipofectamine 3000 (Invi- lowed by Alexa Fluor 488–conjugated secondary antibody trogen). Twenty-four hours posttransfection, WJ-MSC and mounted with DAPI containing mounting media as were induced for transdifferentiation in EGM-2 medium previously described (18). The role of BMP2 on nuclear intheabsence/presenceofBMP2and/orDMH1at37°Cfor translocation of inhibitor of differentiation 1 (ID1) in 72 h. Cells were washed and lysed followed by detection of WJ-MSC and iEC-D7 was separately analyzed by immu- luminescence according to the manufacturer’sprotocol nostaining using confocal microscopy as previously de- (Thermo Scientific) using a multimode reader (Perkin- scribed (18). Elmer) as described previously (23). Similarly, cells were transfected with pMCS-Green Renilla Luc vector subcl- Promoter Gene Cloning Studies oned with the desired promoter region of KDR and SFRP4 In silico analysis of the promoter region of Id1 depicted the and internal control vector, pCMV-Red FireflyLuc,along presence of three putative SMAD-complex binding sites with pCMV-TWIST1-Flag overexpression and/or silencing (BS). Three constructs of the Id1 promoter region were piLenti-TWIST1-siRNA-GFP vectors and/or their respective designed and cloned from WJ-MSC DNA using human controls to determine the role of TWIST1 in the promoter- gene–specific primer sequences containing restriction sites reporter activity of KDR and SFRP4 as described above (23). (XhoI, BamHI, and HindIII; New England Biolabs) by PCR amplification, which were termed Wt-Id1, 2,316 base pairs Immunoblot and Coimmunoprecipitation Analysis Δ Δ (Wt-Id1-bp); BS1-Id1-1,131 bp; BS1-S2 Id1-618 bp; and Cellular protein extracted from WJ-MSC and iEC was sub- Δ fi BS1-S2-S3 Id1-318 bp. These ampli ed constructs were jected to immunoblotting with primary antibodies against subjected to restriction digestion followed by DNA ligation inhibitor of differentiation 1 (ID1), twist family BHLH (T4 DNA Ligase; Invitrogen) into the pMCS-Green Renilla transcription Factor 1 (TWIST1), kinase insert domain fi Luc plasmid constructs (Thermo Scienti c) (23). Similarly, receptor (KDR), secreted frizzled receptor protein (SFRP4), in silico analysis of Kdr promoter up to 5 kb upstream of and b-tubulin as previously described (25). Separately, cell fi transcription start site con rmedthepresenceoftwoen- extracts of WJ-MSC and iEC were coimmunoprecipitated hancer-box (E-box) sequences (TWIST1 BS). Three constructs with ID1 antibody and immunoblotted with TWIST1 anti- of KDR promoter region were designed containing either two body (Abcam) as previously described (23). TWIST1 BS (KDR WT-1,560 bp), one BS (KDR ΔBS1-1,338 bp), or none (KDR ΔBS1-S2-721 bp) and were successfully cloned Chromatin Immunoprecipitation Assay using restriction enzymes (XhoI and BamHI)inpMCS-Green For confirmation of the direct binding of TWIST1 on KDR Renilla Luc vector as described above (23). Also, four E-box and SFRP4 promoter region during transdifferentiation, sequences on the Sfrp4 promoter region up to 5 kb upstream WJ-MSC were transfected with pCMV-TWIST1-Flag and/or of transcription start site were identified. These four con- pCMV-EGFP-N1 and subjected to transdifferentiation. Cells structs, SFRP4-WT-2,097 bp, SFRP4-ΔBS1-1,881 bp, SFRP4- were then washed and cross-linked with 1% formaldehyde ΔBS1-S2-1,668 bp, and SFRP4-ΔBS1-S2-S3-1,341 bp, were followed by lysis using Bioruptor (Diagenode) for 20 cycles cloned using restriction enzymes (XhoI and BamHI)and to get fragmented DNA. The supernatant was collected and T4 DNA ligase into the pMCS-Green Renilla Luc plasmid subjected to immunoprecipitation using the anti-TWIST1 as previously described (23). antibody according to the manufacturer’sprotocol.Thepuri- fied DNA was used as a template for qPCR analysis with Twist1 Overexpression and Silencing primers specific for Twist1-binding sequences on KDR and For overexpression of TWIST1, WJ-MSC were transfected SFRP4 promoter as previously described (25,26). with pCMV-TWIST1-Flag (OriGene) and/or control vector, pCMV-EGFP-N1 (Addgene), using Xfect (Clontech). Con- Flow Cytometry Analysis currently, for silencing of the TWIST1 expression, WJ-MSC WJ-MSCandiECtransfectedwithpCMV-GFP vector were similarly transfected with piLenti-TWIST1-siRNA-GFP were fixed with 4% paraformaldehyde and incubated diabetes.diabetesjournals.org Kaushik and Das 1235 with KDR-phycoerythrin or SFRP4-phycoerythrin along (pLVX-AcGFP/piLenti-Scr-shRNA-GFP) as wound controls with GFP-FITC antibody to evaluate the percentage of or negative control (piLenti-TWIST1-shRNA-GFP)were WJ-MSC and iEC positive for these EC markers using flow transplanted intradermally onto the wounds (1 3 106 cytometry (BD FACSCanto II), and data were analyzed cells/wound) as previously described (25,29,30). Ani- using BD FACSDiva software as previously described (18). mal experimentation and biosafety protocols were ap- proved by the institutional animal ethics committee Generation of Stable rEC With Twist1 Overexpression (approval no. IICT/IAEC/49/2018) and institutional bio- and/or Silencing safety committee (approval no. IICT/IBSC/05/2018). The TWIST1 gene insert (610 bp) was subcloned from pCMV- rate of wound closure was determined using morphometric TWIST1-Flag plasmid vector into pLVX-AcGFP-C1, lentivi- and histological analysis as previously described (18). The ral vector, as described in Supplementary Material. Lentiviral wound sections were subjected to hematoxylin-eosin and particles were prepared by transfecting pLVX-AcGFP-C1/ Sirius red staining to evaluate the reepithelialization, gran- pLVX-AcGFP-TWIST1/piLenti-Scr-shRNA-GFP or piLenti-TWIST1- ulated tissue formation, and collagen deposition (25,29). shRNA-GFP lentiviral plasmid along with second-generation Further, engraftment and paracrine effect of transplanted packaging plasmids: psPAX2 and pMD2.G in HEK-293T cells cells were determined by qPCR analysis of human-specific using Lipofectamine 3000 (Invitrogen). Lentiviral particles and mouse-specific endothelial marker expression, respec- fi were collected and ltered as previously described (18). tively, in the regenerated wound tissues of type 1 and type Next, WJ-MSC were transduced with lentiviral particles 2 diabetic mice as mentioned previously (18,29). Separately, m and subjected for puromycin (0.5 g/mL) selection. The wound tissue sections were subjected to immunostaining GFP-labeled WJ-MSC were subjected to transdifferentiation with TWIST1 and GFP along with human-specific KDR and in EBM-2 medium for 7 and 14 days to generate reprog- mouse/human-specific SFRP4 antibody for determining the — — rammed EC rEC-D7 and rEC-D14, respectively and uti- quantifiable colocalization (Pearson coefficient) (25,29). lized for further in vitro experiments: DiI Ac-LDL uptake, qPCR analysis, flow cytometry analysis, and immunofluo- Evaluation of Neovascularization by Laser Doppler rescence microscopy for deciphering TWIST1-mediated Flowmetry and Confocal Microscopy KDR and SFRP4 gene expression at RNA and protein levels Mice were anesthetized and placed under the laser Doppler as well as for transplantation studies in vivo (18,25). instrument (LDI2-IR; Moor Instruments, Axminster, U.K.) to determine the microcirculatory blood flow in regenerated Physiological Assays wound area. The results were depicted as mean flux WJ-MSC and rEC (pLVX-AcGFP-TWIST1) and control iEC intensity, which was calculated by averaging flux intensity (pLVX-AcGFP-C1/piLenti-Scr-shRNA-GFP) and negative con- of each wound per mice group in both type 1 and type trol (piLenti-TWIST1-shRNA-GFP)atday7or14wereplated 2 diabetic mice (31). Also, the regenerated wound tissue at a cell density of 5 3 103 cells/well to evaluate Twist1- sections were evaluated for the colocalization of human- mediated effect on proliferation potential using BrdU in- specific endothelial markers such as CD31 (platelet en- corporation assay (25). These cells were separately plated at 3 3 – dothelial cell adhesion molecule [PE-CAM]) with GFP to a density of 1 10 cells/well in the Boyden chamber determine the fate of the transplanted rEC or iEC using chemotaxis assay with cells at the upper chamber and VEGF-A confocal microscopy (25). (10 ng/mL; Invitrogen) as chemoattractant in the lower chamber as previously described to determine the role of Statistical Analysis Twist1 on cellular migration (25). Next, these cells were Data represented as mean 6 SEM from experiments that transplanted on the top of Chick chorioallantoic mem- were performed at least thrice. For assessment of the brane (CAM) as previously described (18) to evaluate the difference between groups and their respective controls, effect of Twist1 on the number and length of blood vessel statistical significance was evaluated using one-way or formation using angiocount software (27). two-way ANOVA followed by appropriate analysis such as post hoc, etc., or Student paired t test (Prism, version In Vivo Full Excisional Splint Wound Healing Model Generation in Diabetic Mice 6.05; GraphPad). Photomicrographs and blots from experi- ments were reproduced at least thrice with similar results. Type 1 and type 2 diabetic mice were used to evaluate the in vivo fate of transplanted human rEC and/or iEC. Type Data and Resource Availability 1 diabetes was generated in male or female C57bL/6J mice All data generated or analyzed during this study are included by administrating streptozotocin at a dose of 70 mg/kg here in the published article (and Supplementary Material). intraperitoneally for five consecutive days, whereas, for type 2 diabetes, transgenic db/db mice were used (28). Diabetes generation was confirmed by monitoring blood RESULTS glucose levels at regular intervals for 2 weeks. Both type In Silico Analysis of Microarray Data Sets 1 and 2 diabetic mice were grouped separately and used for We retrieved raw mRNA expression data files of E-MEXP- transplantation studies. Stable human rEC (pLVX-AcGFP- 3071, GSE37045, GSE12891, and GSE25979 from ArrayEx- TWIST1) and/or iEC expressing different control vectors press and NCBI-GEO and performed the quality check followed 1236 Reprogramming in MSC Drives Fate Toward rEC Diabetes Volume 69, June 2020 by normalization of the data sets as described in the workflow basic helix-loop-helix (bHLH) transcription factor fam- (Supplementary Fig. 1A). Next, principal component analysis ily (32) that, in turn, may transcriptionally upregulate of the data sets revealed the gene expression profile to be the angiogenic gene, KDR (Fig. 1B), and downregulate homogenous among the similar cell types but significantly the antiangiogenic gene, SFRP4 (Fig. 1C). distinct among the different cell types (Supplementary Fig. 1B). DEGs in the data sets with a log fold change $2, BMP2-Mediated Effect on Transdifferentiation of identified using the limma package based on their adjusted P WJ-MSC Into iEC values #0.05, depicted 271, 1,107, and 546 upregulated and Transdifferentiation of WJ-MSC into iEC-D7 in presence 336, 871, and 515 downregulated genes in MSC, EPC, and of BMP2 led to a marked increase in DiI Ac-LDL uptake HUVEC, respectively (Supplementary Fig. 1C). These DEGs compared with control iEC, which was perturbed in pres- from each array were classified based on their role in the ence of its receptor inhibitor, DMH1, thereby suggesting different cell signaling pathways using PANTHER, resulting a plausible role of BMP2 in this process (Fig. 2A). Further, in the identification of 983 genes out of 1,554 pathway hits qPCR analysis of endothelial-specific markers depicted spanning 90 PANTHER pathways that are differentially asignificant increase in mRNA expression of CD31, VWF, expressed in all the three data sets (Supplementary Fig. VE-Cadherin, KDR,andeNOS by more than ;3.5 fold in 1C). Among these, 30 pathways relevant to the differ- BMP2-mediated transdifferentiated iEC compared with entiation process were selected that yielded a heat map control, WJ-MSC, which was mitigated in presence of of 265 genes depicting a consistent increase or decrease DMH1 (Fig. 2B). A differential expression pattern of other in their expression levels exhibiting a hierarchical re- endothelial-specific genes, ANGPT2, NRP1, NRP2, TIE2,etc., lationship between MSC, late endothelial progenitor cell was observed in these treated iEC (Fig. 2C), thus suggest- (lEPC), and HUVEC (Supplementary Fig. 2A). Next, self- ing modulation of selective endothelial-specificgeneex- organizing map clustering of these 256 genes depending pression by BMP2. upon the expression pattern resulted in selection of clusters 11, 16, and 21 that depicted consistent increase BMP2-Mediated SMAD-Induced Transcriptional but clusters 15, 20, and 25 with a consistent decrease in gene Regulation of Id1 expression profilefromMSCtolEPCandlEPCtoHUVEC BMP2 binds to its receptors, BMPRI and BMPRII, and (Supplementary Fig. 2B). Lastly, these clusters consisting of activates its downstream signaling via sequential SMAD 72 genes were represented using a heat map that depicted activation to regulate its target genes (33). Interestingly, a consistent increase or decrease in expression in MSC to literature also suggests tBMP2-mediated regulation of its lEPC and lEPC to mature EC, HUVEC (Fig. 1A). downstream effectors in a SMAD-independent manner (34). Thus, it becomes pertinent to decipher the downstream In Vitro Validation of In Silico Data Sets During MSC signaling pathway of BMP2 during transdifferentiation of Transdifferentiation Into Endothelial Lineage Cells WJ-MSC into iEC to be SMAD dependent or independent. A random selection of 41 out of 72 genes from in silico data To evaluate this, we determined the global gene expres- sets was validated using qPCR analysis in the prior well- sion profile of all the SMADs along with BMP2 receptors characterized WJ-MSC and transdifferentiated iEC-D7 and in WJ-MSC and iEC in the absence or presence of BMP2. iEC-D14 (18). The results of in vitro studies depicted an BMP2 could significantly increase Smad2 expression in increased expression of BMP1, BMP2, KDR, SERPINE1, WJ-MSC, while in iEC, BMP2 treatment led to a signifi- RHOB1, IFNGR1, SMAD1, IL1A, BIRC5, ITGB3,andRGS5 cant increase, by four to fivefold, in SMAD1, SMAD4, and decreased expression of ID1, SFRP4,andBIRC3 (Fig. SMAD5,andSMAD8 as well as inhibitory SMADs—SMAD6 1B–E), which was corroborated well with the in silico (;s7-fold) and SMAD7 (;15-fold)—suggesting the occur- observations and thereby incited us to identify the plau- rence of SMAD-dependent signaling downstream to BMP2 sible molecular targets that can modulate the transdiffer- (Fig. 2D). Literature suggests various BMP2-mediated SMAD- entiation of WJ-MSC into iEC. Next, the protein-protein regulated target genes such as ID1,whichisknowntoinhibit interaction analysis of these identified genes using STRING cell differentiation (32). BMP2 treatment led to a significant v9.1 online software tool displayed regulatory connections increase in ID1 expression in WJ-MSC (Fig. 3A). In contrast, among them suggesting cross talk between different path- a decreased expression of ID1 was also observed during ways, as genes participating in one pathway were observed transdifferentiation of WJ-MSC into iEC that were further to regulate the expression of other genes by their activation decreased in presence of BMP2 (Fig. 3A), which led us to or deactivation in other pathways (Supplementary Fig. 3). explore the transcriptional regulation of ID1 by BMP2. The Among these, during transdifferentiation of WJ-MSC into presence of four SMAD complex BS was identified on the iEC, a consistent eightfold and ;twofold high expression of promoter region of ID1 (Supplementary Fig. 4A), which the growth factor BMP2 (Fig. 1C) and its downstream were cloned in pMCS-Green Renilla Luc vector by sequential effector, Smad1 (Fig. 1D), suggests a Smad-mediated deletion of BS (Supplementary Fig. 4B). Next, promoter- regulation of an atypical transcription factor, ID1. ID1 reporter luciferase activity was determined as described in lacks the DNA-binding domain and regulates its down- RESEARCH DESIGN AND METHODS. The results depicted a signifi- stream effector genes via its cofactors belonging to the cant decrease in the promoter activity of the Wt-ID1 diabetes.diabetesjournals.org Kaushik and Das 1237

Figure 1—Differential gene expression profiles in MSC, endothelial progenitors, and mature EC. A: A heat map depicting 72 genes selected from microarray data sets with an increase/decrease in expression profile in MSC, late EPCs, and mature EC, with HUVEC as reference. B–E: In vitro validation of randomly selected 41-gene in silico analysis of gene expression profile retrieved from the microarray data sets using qPCR in cultured WJ-MSC and transdifferentiated iEC for day 7 (iEC-D7, early EPC) and 14 (iEC-D14, late EPC). Statistical significance with *P # 0.05 as compared with WJ-MSC. promoter construct in iEC compared with WJ-MSC (Fig. to act as a negative transcriptional regulator of its target 3B). As expected, treatment with BMP2 further led to genes, whereas HIF-2a has a proven role under hypoxic adecreaseintheluciferaseactivity in iEC but not in conditions. However, TWIST1 has been reported in the liter- WJ-MSC. The sequential deletion of BS1 led to a further ature to act both as a negative and a positive regulator of its increase in promoter activity in WJ-MSC as well as iEC target gene transcription (35,36). Excitingly, a concomitant compared with the Wt-ID1 group, suggesting that the BS1 increase in TWIST1 expression with decreased levels of ID1 in site is essential for the negative regulation of ID1 during iEC (Fig. 3C) suggested a negative correlation between ID1 and WJ-MSC transdifferentiation into iEC. However, there was TWIST1-mediated regulation of its downstream effectors no significant change in promoter activity after the deletion during WJ-MSC transdifferentiation into iEC. Coimmunopre- of both BS1 and BS2 by the presence of BS3 and the deletion cipitation of WJ-MSC and iEC with ID1 and immunoblot with of BS1, BS2, and BS3 by the presence of BS19.Thesedata TWIST1showedbindingofID1withTWIST1inWJ-MSCbut suggest that BMP2-mediated increased expression of inhib- not in the transdifferentiated iEC (Fig. 3D), suggesting a de- itory SMADs—SMAD6 and SMAD7—downregulates the creased expression of ID1 in iEC renders the TWIST1 free for ID1 transcription. Next, immunoblot analysis depicted its downstream transcriptional regulation of its target an increased protein expression of ID1 in WJ-MSC only genes. However, in WJ-MSC, ID1 sequesters TWIST1 but not in iEC in the presence of BMP2 treatment that was and thereby makes it unavailable for its downstream mitigated by DMH1 (Fig. 3C). As mentioned earlier, ID1 regulation. ID1 is a nuclear protein, and immunofluores- imparts downstream regulation by interacting with the cent staining revealed that BMP2 induced marked in- bHLH family of transcription factors such as HEY1, HIF- crease in localization of ID1 in the nucleus of WJ-MSC 2a, and/or TWIST1, which may regulate the expression of but not iEC, an effect that was perturbed by DMH1 target genes, KDR and SFRP4. Among these, HEY1 is known (Fig. 3E). Additionally, TWIST1 mRNA expression was 1238 Reprogramming in MSC Drives Fate Toward rEC Diabetes Volume 69, June 2020

Figure 2—BMP2 potentiates WJ-MSC transdifferentiation into endothelial lineage cells via differential expression of SMADs. A: Repre- sentative images of higher DiI Ac-LDL staining in iEC transdifferentiated from WJ-MSC in the presence of BMP2, which was perturbed by DMH1 (BMP2 receptor inhibitor) compared with WJ-MSC. Graphs depicting differential mRNA expression profile of endothelial markers (B and C) and regulatory and inhibitory Smads (D) in iEC and WJ-MSC treated with BMP2 in the absence or presence of DMH1. P # 0.05 compared with *WJ-MSC, #iEC, $iEC1BMP2.

observed to be significantly increased during the trans- the tags Flag or GFP, KDR, SFRP4, and TWIST1 in trans- differentiation of WJ-MSC into iEC-D7 and iEC-D14 fected WJ-MSC (Supplementary Fig. 4F). As described (Supplementary Fig. 4C). Next, Western blot analysis in RESEARCH DESIGN AND METHODS, the presence of putative also depicted a marked increase in TWIST1 and KDR TWIST1 binding sites on the promoter region of identified but a decrease in SFRP4 expression in iEC-D7 and iEC- target genes KDR (Supplementary Fig. 5A and B)andSFRP4 D14 as compared with WJ-MSC (Supplementary Fig. 4D). (Supplementary Fig. 5C and D) led us to evaluate the Also, flow cytometry analysis depicted high KDR and low promoter-reporter assay. A significant increase in Wt-KDR SFRP4 in iEC compared with WJ-MSC (Supplementary Fig. promoter activity was observed in iEC by 10-fold in the 4E). These data provide critical insights into the role of presence of TWIST1 overexpression (Supplementary Fig. 6A). TWIST1 as a downstream transcriptional modulator of the Further, a sequential deletion of BS1 (DBS1-KDR)resultedin BMP2-ID1 signaling pathway during transdifferentiation of a fivefold increase in KDR promoter activity compared with WJ-MSC into iEC. control vector–transfected iEC groups (Supplementary Fig. 6A), suggesting TWIST1 positively regulates KDR promoter TWIST1 Transcriptionally Regulates KDR and SFRP4 activity from two distinct binding sites. Similiarly, TWIST1 Expression during Transdifferentiation overexpression led to a significant increase in luciferase To further evaluate the TWIST1-mediated transcriptional activity in WJ-MSC by ;twofold, which was far less compared regulation of KDR and SFRP4 during the transdifferentia- to iEC (Supplementary Fig. 6B). This effect was significantly tion process, we used the gain- and loss-of-function ap- perturbed in the presence of TWIST1 silencing in both iEC proach by overexpression and/or silencing of TWIST1 gene (Supplementary Fig. 6A) and WJ-MSC (Supplementary Fig. that was confirmed by immunoblotting the expression of 6B). Furthermore, the deletion of both the binding sites diabetes.diabetesjournals.org Kaushik and Das 1239

Figure 3—BMP2-mediated coregulation of ID1 and TWIST1 during transdifferentiation of WJ-MSC into iEC. A: ID1 mRNA expression in WJ-MSC and iEC showed a significant increase in its expression in BMP2-treated samples in the absence of DMH1 in WJ-MSC only. In iEC, BMP2 treatment further decreased ID1 expression. B:Promoterreporter(Pr) luciferase assay revealing decreased ID1 promoter activity in the presence of Wt-ID1 in iEC as compared with WJ-MSC. BMP2 treatment further increased and decreased promoter activity in WJ-MSC and iEC, respectively, while DMH1 reversed the BMP2 effect. Sequential deletion of BS2, BS19, and BS3 did not result in any significant change, whereas BS1 deletion led to an increased promoter activity, suggesting the inhibitory role of BS1 in regulating SMAD- induced transcriptional regulation of Id1 during transdifferentiation of WJ-MSC into iEC (P # 0.05 compared with *WJ-MSC, #iEC). C: Western blot analysis depicted a simultaneous decrease and increase in ID1 and TWIST1 expression, respectively, in iEC as compared with WJ-MSC, while BMP2-mediated induction of ID1 and TWIST1 was observed only in WJ-MSC and iEC, respectively—an effect that was mitigated by DMH1, thereby suggesting that TWIST1 induction has a positive role during WJ-MSC transdifferentiation into iEC. D: Coimmunoprecipitation of WJ-MSC and iEC with ID1 and immunoblot with TWIST1 depicted binding of ID1 with TWIST1 in WJ-MSC but not in iEC. E: Immunostained confocal images of WJ-MSC and iEC depicting a marked increase in the expression of ID1 within the nucleus in WJ-MSC but not iEC in the presence of BMP2 that was perturbed by DMH1 (Scale bar, 20 mm). IB, immunblot; IP, immunoprecipitation; Mol Wt, molecular weight; VC, vehicle control.

significantly abolished the promoter activity (Supplemen- was also observed in WJ-MSC but with lesser fold change tary Fig. 6A and B). Next, promoter-reporter assay for the (Supplementary Fig. 6D). Interestingly, the silencing of Wt-SFRP4 promoter depicted a significant decrease in lu- TWIST1 could not regain the SFRP4 promoter activity, ciferase activity in the presence of Twist1 overexpression in suggesting the involvement of other cofactors along with iEC (Supplementary Fig. 6C) as well as in WJ-MSC (Supple- TWIST1 for transcriptional regulation of the SFRP4 gene. mentary Fig. 6D). Interestingly, the sequential deletion of Next, for evaluation of the direct interaction between TWIST1 BS1 (DBS1-SFRP4)ledtoarobustdecreaseinpromoter and its putative binding sites on KDR and SFRP4 promoter, activity in iEC (Supplementary Fig. 6C). However, subsequent chromatin immunoprecipitation analysis was performed that deletion of both BS1 and BS2 (DBS1-S2-SFRP4)andBS1, depicted an efficient differential binding of TWIST1 at binding BS2, and BS3 (DBS1-S2-S3-SFRP4) did not show any sig- sites—BS2 on KDR promoter (Supplementary Fig. 7A)but nificant change in promoter activity, suggesting BS1 and both BS1 and BS2 on SFRP4 promoter (Supplementary Fig. BS2 are essential sites for TWIST1 binding. A similar effect 7B)—thereby correlating well with the promoter-reporter 1240 Reprogramming in MSC Drives Fate Toward rEC Diabetes Volume 69, June 2020

Figure 4—Transplantation of TWIST1 rEC–enhanced neovascularization-mediated enhanced rate of wound closure. Laser Doppler images of type 1 (upper panel) and type 2 (lower panel) diabetic mice groups (A) and quantification of percent mean flux intensity (B) depicting higher microcirculatory blood flow in the TWIST1 rEC–transplanted regenerated wounds of type 1 (above) and 2 (below) diabetic mice as compared with their respective control and/or TWIST1-knockdown iEC–transplanted groups (*P # 0.05 compared with pLVX-AcGFP-iEC Tx group). C: Representative images of morphometrical analysis of type 1 (upper panel) and type 2 (lower panel) diabetic mice wounds postsurgery day 14 (PS-D14). (Dotted line representing the remnant wound periphery at postsurgery day 14, outer ring depicting the inner edge of the splint, and scale bar, 5 mm, representing the initial wound size (PS-D0). D: Graph depicting an increased rate of wound closure in TWIST1 rEC– transplanted type 1 (left panel) and 2 (right panel) diabetic mice as compared with other control empty vector/Scr shRNA/negative control (TWIST1 knockdown) iEC–transplanted mice group. Scr, scrambled; Tx, transplanted. P # 0.05 compared with *wound control–no Tx group, #pLVX-AcGFP-iEC Tx group. luciferase activity. These data suggest that TWIST1 plays depicted a significant increase and decrease in TWIST1 a crucial role by transcriptional regulation of KDR and in part expression in rEC at days 7 and 14 transduced with of SFRP4 by promoting KDR while inhibiting SFRP4 expres- TWIST1 overexpression and silencing (negative control) sion during transdifferentiation of WJ-MSC into iEC. lentiviral particles, respectively (Supplementary Fig. 8B). Immunofluorescence microscopy depicted GFP expres- TWIST1 Reprogramming of WJ-MSC Enhances the sion in all the transduced cell groups, while coimmunos- Endothelial Lineage Commitment taining of GFP and DAPI confirmed the nuclear localization of Next, to evaluate the role of TWIST1 in determining the overexpressed TWIST1, a transcription factor, as compared fate of WJ-MSC toward endothelial transdifferentiation, with empty vector control– and piLenti-TWIST1-shRNA-GFP– TWIST1 was subcloned in a lentiviral vector as described in transduced iEC (negative control) groups (Supplementary Fig. RESEARCH DESIGN AND METHODS. WJ-MSC were transduced 8C). TWIST1 overexpression led to an increase in KDR ex- with TWIST1 overexpressing/silencing lentiviral particles pression (;85%) and a decrease in SFRP4 expression (;35%), followed by puromycin selection and subjected for endo- while its silencing reversed this phenomenon in TWIST1 rEC thelial differentiation of these rEC. TWIST1 overexpres- as analyzed using flow cytometry assay (Supplementary Fig. sion in rEC was confirmed by immunoblotting that depicted 8D). To substantiate the endothelial transdifferentiation a shift in molecular weight of TWIST1 (27 kDa) fused with of our rEC, we carried out various physiological and GFP (27 kDa) gene to 54 kDa as compared with GFP alone molecular assays to confirmtheroleofTWIST1,which (Supplementary Fig. 8A). Next, the qPCR analysis also has not yet been implicated in similar cell fate determination diabetes.diabetesjournals.org Kaushik and Das 1241

Figure 5—Histological analysis of regenerated wound. Representative images of hematoxylin and eosin staining (A) and quantitation (B) and Sirius red staining (C) and quantitation (D) of stained cells depicting higher granulation and epidermal thickness in TWIST1 rEC–transplanted group compared with control vector/Scr shRNA/TWIST1-knockdown iEC–transplanted groups suggesting skin tissue regeneration at the wound bed. Similarly, representative images of Sirius red staining (C) and quantification (D) of the number of stained cells depicting higher collagen deposition in the TWIST1 rEC–transplanted group compared with control iEC–transplanted groups. (Horizontal arrow, wound gap; vertical arrow, epidermal thickness). Scr, scrambled; Tx, transplanted. P # 0.05 compared with *pLVX-AcGFP-iEC Tx group, #piLenti-Scr- shRNA-GFP-iEC Tx group. to the best of our knowledge. The rEC showed a marked by decreased expression of KDR compared with control increase in DiI Ac-LDL uptake as compared with control iEC, iEC (Supplementary Fig. 10A). Similarly, SFRP4 expres- which was further perturbed in iEC with TWIST1 knockdown sion was decreased in rEC compared with WJ-MSC (con- (negative control) as compared with WJ-MSC (control) at day trol), but there was an increase in SFRP4 expression when 7 (data not shown) and day 14 (Supplementary Fig. 9A). TWIST1 was silenced (Supplementary Fig. 10B), confirm- TWIST1-mediated reprogramming of WJ-MSC into rEC was ing that TWIST1 negatively regulates SFRP4 during trans- further confirmed by qPCR analysis of the expression profile differentiation of WJ-MSC into rEC. These data indicate of endothelial marker genes. Our results indicated a signifi- that TWIST1 reprogramming resulted in enhanced endo- cant increase in expression of several endothelial markers thelial lineage transdifferentiation of WJ-MSC. such as KDR, VEGFR1, VE-cadherin, ANGPT2, eNOS, VWF, CD31, EDN1, NRP1, TIE-2,andCD146 in rEC-D14, whereas TWIST1 Reprogramming Enhanced the Physiological there was a significant increase in expression of SFRP4 upon Functions of rEC TWIST1 silencing in iEC (Supplementary Fig. 9B and C). This The functional activation of rEC was determined by eval- observation was further supported by immunofluores- uating cell proliferation and migration assays after 7 and cent staining of these cells, which depicted a marked 14 days of reprogramming. There was a significant de- increase in KDR expression in rEC and iEC at day 14 as crease in proliferation potential of rEC compared with compared with WJ-MSC (control), whereas TWIST1 knock- WJ-MSC at days 7 and 14 (Supplementary Fig. 11A), thereby down perturbed the transdifferentiation of WJ-MSC into iEC confirming the transdifferentiation of cells during which 1242 Reprogramming in MSC Drives Fate Toward rEC Diabetes Volume 69, June 2020

Figure 6—Enhanced engraftment of TWIST1 rEC in regenerated type 2 diabetic mouse wound bed. Representative confocal images (A) and quantitation (B) of colocalization of TWIST1 and GFP depicting increased engraftment in the TWIST1 rEC–transplanted group. C: qPCR analysis revealing enhanced human-specific endothelial marker gene expression in regenerated skin tissue of type 2 diabetic mice wounds suggesting direct incorporation of transplanted TWIST1 rEC. D: Significant increase in mouse gene–specific endothelial markers in the TWIST1 rEC–transplanted group suggesting enhanced neovascularization occurring in a paracrine manner by the transplanted human TWIST1 rEC in regenerated wounds as compared with control vector/Scr shRNA/TWIST1-knockdown iEC–transplanted groups. Scr, scrambled; Tx, transplanted. *P # 0.05 compared with the pLVX-AcGFP-iEC Tx group.

proliferation is halted as expected. The Boyden chamber– Transplantation of TWIST1 rEC Potentiated Vascularity chemotaxis assay revealed a significant enhancement of at the Diabetic Wound Bed migratory potential in rEC as compared with WJ-MSC that To evaluate the in vivo vasculogenic potential of trans- were perturbed by TWIST1 silencing in iEC after 7 and 14 days planted TWIST1 rEC, full-thickness excisional splint wound of transdifferentiation (Supplementary Fig. 11B). As late EPC healing model was generated in streptozotocin-induced type is known to incorporate into new blood vessels but not the 1 (Supplementary Fig. 12A)aswellasdb/db type 2 diabetic early EPC, we evaluated the vasculogenic potential of iEC-D14 mice (Supplementary Fig. 12B). The stable TWIST1 rEC and/ by CAM angiogenic assay. Stable rEC or iEC at day 14 were or iEC (1 3 106 cells/wound) were transplanted intradermally mixed with matrigel and loaded on the top of CAM as at the periphery of the wound. Laser Doppler flowmetry was described in RESEARCH DESIGN AND METHODS. After incubation of performed to evaluate the microcirculatory blood flow in 72 h, the morphological analysis of matrigel depicted a higher regenerated wounds. The results showed a higher mean flux vasculogenic potential of rEC as compared with control iEC intensity (.300) in the wounds transplanted with rEC (TWIST1 with empty vector/scrambledsiRNA,aswellasiECwith overexpressing) as compared with other positive (vector con- TWIST1 knockdown (Supplementary Fig. 11C). Together, trols) and negative (TWIST1 silencing) control iEC–transplanted these observations suggested that rEC with enhanced type 1 and 2 diabetic mice groups (Fig. 4A). Quantitation of the vasculogenic capacity can be of therapeutic potential for percent mean flux intensity depicted a significant increase in hypovascularity-associated diseases like chronic nonhealing di- the TWIST1 rEC–transplanted group as compared with iEC- abetic wounds. transplanted groups (Fig. 4B), suggesting enhanced vascularity diabetes.diabetesjournals.org Kaushik and Das 1243

Figure 7—TWIST1 rEC transplantation regulates KDR and SFRP4 expression in the regenerated wound tissue of type 2 diabetic mice. Representative coimmunostaining confocal images of regenerated wound tissue depicted a higher colocalization of GFP/KDR (A) and quantification of colocalization (B) and lower colocalization of GFP/SFRP4 (C) and colocalization quantification (D) in TWIST1 rEC– transplanted type 2 diabetic mouse group as compared with other iEC-transplanted mouse groups, suggesting higher neovascularization at the regenerated wounds. Tx, transplanted. *P # 0.05 compared with the pLVX-AcGFP-iEC Tx group.

at the wound bed of type 1 and 2 diabetic mice transplanted 5D)inTWIST1-silenced iEC–transplanted (negative control) as with TWIST1-reprogrammed iEC. compared with iEC-transplanted (control vector) type 1 (data not shown) and type 2 diabetic mice groups. Transplantation of TWIST1 rEC Accelerated Wound Closure in Full Excisional Splinted Diabetic Wounds Enhanced Engraftment of TWIST1 rEC Potentiates The morphometric analysis of regenerated wound on post- Neovascularization in Regenerated Diabetic Wounds surgery day 14 depicted an enhanced wound healing in rEC- For evaluation of the engraftment of transplanted cells, transplanted type 1 and 2 diabetic mice groups as compared coimmunostaining of GFP with TWIST1 was evaluated by with other groups (Fig. 4C). The rate of wound closure was confocal microscopy, which depicted a higher colocaliza- ;78% and 84% in Twist1 rEC–transplanted type 1 and tion of TWIST1 with GFP in TWIST1 rEC–transplanted 2 diabetic mice groups, respectively, as compared with other type 1 (Supplementary Fig. 13A) and type 2 (Fig. 6A)diabetic iEC control or negative control groups (Fig. 4D). This was mice group as compared with iEC-transplanted (control vector) further corroborated by histological analysis of regenerated groups (quantitative analysis) (Fig. 6B and Supplementary Fig. wounds by hematoxylin-eosin (Fig. 5A and B) and collagen 13B). The higher engraftment of TWIST1 rEC in regenerated staining (Fig. 5C and D), which revealed an increased wounds was further supported by qPCR analysis of human- granulation and collagen deposition in the TWIST1 rEC– specific endothelial marker genes—VEGFR2 (KDR), CD31, transplanted group as compared with control iEC–transplanted TIE-2, VWF, VE-Cadherin,andNRP2—suggesting direct en- groups. Quantitative analysis depicted no significant change in graftment of rEC at the wound bed leading to increased reepithelialization as evident from hematoxylin-eosin staining neovascularization in the type 1 (Supplementary Fig. 13C) (Fig. 5B)butasignificant decrease in collagen deposition (Fig. and type 2 (Fig. 6C) diabetic wounds. To determine whether 1244 Reprogramming in MSC Drives Fate Toward rEC Diabetes Volume 69, June 2020

Figure 8—Enhanced neovascularization by transplantation of the TWIST1 rEC–transplanted group at the wound bed of type 2 diabetic wounds. Higher coimmunostaining of GFP and human-specific CD31 (A) and quantification of colocalization (B) in type 2 diabetic mice confirmed the fate of the transplanted TWIST1 rEC toward increased neovascularization at the wound site of the nonhealing diabetic wounds. This observation was further supported by an increased coimmunostaining of a-SMA and CD31, suggesting the generation of mature vessels. #/*P # 0.05 compared with the pLVX-AcGFP-iEC Tx group. the transplanted TWIST1 rEC regulate wound microenviron- diabetic mice. An increased colocalization of GFP with ment in a paracrine fashion, we performed qPCR analysis for SFRP4 in the type 1 (Supplementary Fig. 14D)andtype mouse-specific endothelial marker genes. CD31, Tie-2, vWF, 2(Fig.7D)diabeticwoundswasevidentfromthequan- VE-Cadh,andNrp2 were also observed to be higher in TWIST1 tified images of TWIST1-silenced iEC–transplanted groups rEC–transplanted type 1 (Supplementary Fig. 13D)andtype as compared with control groups, thereby corroborating 2(Fig.6D) diabetic mice groups as compared with groups with the in vitro observation of TWIST1-mediated tran- transplanted with other control vectors, iEC and/or TWIST1- scriptional upregulation and downregulation of KDR and knockdown iEC (negative control). Next, immunostaining SFRP4 expression, respectively. Finally, TWIST1 rEC trans- with human-specific KDR antibody and GFP revealed plantation–mediated enhanced neovascularization of the di- a higher colocalization at the wound bed of the TWIST1 abetic wounds was determined by immunostaining with rEC–transplanted group compared with control/empty human-specific CD31. A higher CD31 colocalization with vector iEC–transplanted groups in both type 1 (Supple- GFP at the wound of type 1 (Supplementary Fig. 15A) mentary Fig. 14A) and type 2 (Fig. 7A) diabetic mice. (quantification of colocalization) (Supplementary Fig. Quantification of colocalization revealed no significant 15B)andtype2(Fig.8A and B) diabetic mice trans- colocalization of GFP with KDR in the regenerated wound planted with TWIST1 rEC as compared with control bed of type 1 (Supplementary Fig. 14B) and type 2 (Fig. 7B) vector iEC– and/or TWIST1-silenced (negative control) diabetic mice transplanted with TWIST1-silenced iEC as iEC–transplanted groups. Finally, detection of a-SMA compared with scrambled shRNA iEC. Similarly, a de- (mural cells, green) and CD31 (engrafted rEC, red) in creased colocalization of GFP with SFRP4 was observed in regenerated skin tissues in TWIST1 rEC–transplanted type the regenerated wound bed of TWIST1 rEC–transplanted 1 (Supplementary Fig. 15C and D) and type 2 (Fig. 8C and D) type 1 (Supplementary Fig. 14C)andtype2(Fig.7C) diabetic mice depicted potentially higher blood vessel diabetes.diabetesjournals.org Kaushik and Das 1245 maturation (yellow) as compared with other transplanted further induced in the presence of endothelial-specific groups. These observations depictedthefateofthetrans- growth factor–containing medium. As a similar approach planted TWIST1 rEC toward neovascularization for improved to increase the EC repertoire, others in the field have used vascularity-mediated diabetic wound tissue regeneration. different types of stem and somatic cells such as iPSC (5), amniotic cells (10), and fibroblasts (9,12,13,45). Next, DISCUSSION our study identified and used TWIST1, which is a master Neovascularization in hypovascularity-related nonhealing transcriptional regulator of mesodermal development and diabetic wounds can be enhanced by exogenous EC trans- has been reported to promote ocular angiogenesis (46). The plantation therapy to improve the vascularity. However, molecular mechanism of TWIST1 differs during the regu- prior literature suggests that transplantation of mature EC lation of normal and tumor angiogenesis (47). Twist1 fails to improve vascularity as well as functional improve- knockdown adversely affected embryonic vascular growth ment of the injured tissue (37,38). Recent studies on the in Xenopus (48). Singh et al. (49) reported Twist1 over- clinical and therapeutic application of autologous EPC trans- expression in Giant cell tumor stromal cells induced plantation depicted limited improvement in neovasculari- expression of endolethial marker genes such as Vegfa zation and tissue repair (39). A reduced levels as well as and Vegfr1, whereas its knockdown depicted decreased dysfunctional EC with an impaired proliferative, and mo- expression levels of these endothelial marker genes, using bilization capability, integrin profile, low differentiation and in vitro studies. Similarly, we also observed an enhanced incorporation into new blood vessels were observed in Type mRNA expression of endothelial markers such as 1 and Type 2 diabetes (40–42) thereby, limiting the source VEGFR1, VEGFR2, CD31, VWF, TIE-2,andCD146,etc., of EPC to be autologous in origin for transplantation. Thus, in rEC. TWIST1-mediated biological functions are diverse, isolation/generation of EPC or iEC from the repertoire of which is achieved by its homo/heterodimers interac- adult stem cells, with higher proliferative and differentia- tion with other bHLH coregulators such as E2 proteins tion potential toward EC, is warranted to undertake the (35,36,47). It transcriptionally regulates its downstream unmet medical needs. To address that, we attempted to targets by recognizing consensus enhancer-box (E-box) accelerate the process of transdifferentiation of adult hu- sequence “CANNTG” on the promoter region of its target man WJ-MSC into iEC by identifying molecular targets that genes (50). The activation or suppression of Twist1 target can promote the process. In silico analysis led us to identify genes is determined by its stimulatory or inhibitory molecular target BMP2, a growth factor that activates its partner that modulates chromatin modifiers or RNA downstream target ID1 via SMAD1-dependent signaling. polymerase 2 transcription complex formation (35,36,50). BMP2 has been sporadically reported to play a role in tumor Our findings, too, showed TWIST1 as both positive tran- vasculogenesis via SMAD1, ERK1/2–dependent ID1 expres- scriptional regulator of the angiogenic gene VEGFR2 (KDR) sion which enhanced the proliferative and tube forming and negative transcriptional regulator of antiangiogenic gene capability of the cells and its inhibition using a BMP2 SFRP4. antagonist suppressed angiogenesis in tumor cells in vivo MSC have been used for transdifferentiation into en- (34). Similarly, our study revealed that BMP2 activated dothelial lineage cells using growth factors (15–18) and canonical SMAD signaling in WJ-MSC but during trans- anti-inflammatory drugs (18), and their subsequent trans- differentiation into iEC this gets interrupted by the pres- plantation enhanced neovascularization-mediated wound ence of inhibitory SMADs that led to a decreased expression healing in a full excisional splint wound healing model of ID1, a BMP2 target gene, thereby suggesting the in- generated in C57BL/6J mice. Aguilera et al. (8) trans- volvement of coregulation by other transcription factors in differentiated MSC into EC for 14 (EC-D14) and 30 (EC- the downstream signaling. Inhibition of ID1 has also been D30)daysandreportedaninsignificant difference in reported to regulate embryonic stem cell differentiation wound healing profile in mice transplanted with EC-D14 into EC (43). Hetero-dimerization of ID1 is sufficient to or EC-D30. This led us to culture iEC under transdiffer- prevent the binding of bHLH family proteins on DNA entiation up to day 14 for iEC transplantation. Our results (44). It not only blocks the transcription of its target gene clearly describe that transplantation of TWIST1 rEC potenti- but also inhibits the function of the bHLH transcription ated neovascularization at the diabetic wound site by en- factor by sequestration of ubiquitous E proteins (32). Our hanced microcirculatory blood flow and enhanced KDR and in vitro mechanistic studies proved our hypothesis that decreased SFRP4 protein expression in the regenerated skin ID1 sequesters off TWIST1 and inhibits the latter in tran- tissue at the diabetic wound bed. This study suggests use of scriptionally activating its target genes in WJ-MSC, while, transplantation of TWIST1-reprogrammed EC as a plausible during transdifferentiation of WJ-MSC into iEC due to autologous adult stem cell–based therapy for improving activation of inhibitory SMADs, ID1 expression is de- neovascularization in hypovascularity-related diseases or creased and is unavailable to interact with TWIST1, thereby tissue injury. enabling the transcriptional regulation of TWIST1-dependent In conclusion, these observations set the stage for future target genes. Further, to generate proof of concept, we clinical application using small molecule therapeutic approaches generated reprogrammed iEC by stable overexpression of to target this signaling pathway to modulate KDR and SFRP4 Twist1 using a lentiviral vector in WJ-MSC that was or transgenically modifying adult stem cells using CRISPR-Cas9 1246 Reprogramming in MSC Drives Fate Toward rEC Diabetes Volume 69, June 2020 technology instead of lentiviral vectors to generate rEC for 17. Oswald J, Boxberger S, Jørgensen B, et al. Mesenchymal stem cells can be transplantation therapy. differentiated into endothelial cells in vitro. Stem Cells 2004;22:377–384 18. Kaushik K, Das A. Cycloxygenase-2 inhibition potentiates trans-differentiation of Wharton’s jelly-mesenchymal stromal cells into endothelial cells: transplantation – Funding. A.D. acknowledges the funding provided by the Council of Scientific enhances neovascularization-mediated wound repair. Cytotherapy 2019;21:260 and Industrial Research (CSIR), Ministry of Science & Technology, Government of 273 India, for Niche Creating High Science Projects under Healthcare theme: CSIR-IICT 19. Thomas PD, Campbell MJ, Kejariwal A, et al. PANTHER: a library of protein – MLP0052 (PROMPT), MLP0053 (GRAFT). A fellowship provided by UGC-JRF/SRF to families and subfamilies indexed by function. Genome Res 2003;13:2129 2141 K.K. is gratefully acknowledged (manuscript communication number: IICT/Pubs./ 20. Cheng CC, Chang SJ, Chueh YN, et al. Distinct angiogenesis roles and surface 2019/379). markers of early and late endothelial progenitor cells revealed by functional group Duality of Interest. No potential conflicts of interest relevant to this article analyses. BMC Genomics 2013;14:182 were reported. 21. Franceschini A, Szklarczyk D, Frankild S, et al. STRING v9.1: protein-protein Author Contributions. 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