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