The Effect of Fat Grafting on Scars Hyperpigmentation: A Systematic Review and Meta-Analysis.

BACKGROUND: Hyperpigmented scars, particularly in exposed body areas, can be difficult to conceal and may evoke psychological distress. While the precise causes of scar dyschromia are not fully understood, alterations in melanogenic activity appear to hold more significance than changes in melanocyte quantity. Current treatments encompass laser interventions. However, it is essential to consider their costs and potential complications in relation to their limited proven effectiveness. Fat grafting has gained interest as a scar modulation technique due to its regenerative properties, and its efficacy in reducing scar hyperpigmentation is currently under investigation. METHODS: A systematic review and meta-analysis was reported according to PRISMA guidelines. PubMed, Embase, and Cochrane Library databases were accessed. PROSPERO registration number is CRD42023457778. The primary outcome was a change in scar pigmentation after fat grafting. Pigmentation changes after fat grafting were calculated using the standardized mean difference (SMD) between baseline and postoperative scores according to POSAS and VSS scales. Bias assessment was conducted according to the National Institute for Health and Clinical Excellence quality assessment tool. RESULTS: A total of 8 articles meeting inclusion and exclusion criteria were identified, involving 323 patients with hyperpigmented scars treated with fat grafting. A significant difference in scar pigmentation was noted after treatment with fat grafting according to observers' ratings, with a SMD of - 1.09 [95% CI: - 1.32; - 0.85], p<0.01. The SMD for patient-reported scar pigmentation after treatment with fat grafting was - 0.99 [96% CI: - 1.31; - 0.66], p<0.01. Four studies provided objective measurements of melanin changes after fat grafting and revealed inconsistent findings compared to subjective observations. CONCLUSIONS: Fat grafting shows promise in ameliorating hyperpigmented scars based on subjective assessments, but further corroborating evidence from objective measures is required. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Metformin and adipose-derived stem cell combination therapy alleviates radiation-induced skin fibrosis in mice.

BACKGROUND: Radiation therapy often leads to late radiation-induced skin fibrosis (RISF), causing movement impairment and discomfort. We conducted a comprehensive study to assess the effectiveness of metformin and adipose-derived stem cells (ASCs), whether autologous or allogeneic, individually or in combination therapy, in mitigating RISF. METHODS: Using a female C57BL/6J mouse model subjected to hind limb irradiation as a representative RISF model, we evaluated metformin, ASCs, or their combination in two contexts: prophylactic (started on day 1 post-irradiation) and therapeutic (initiated on day 14 post-irradiation, coinciding with fibrosis symptoms). We measured limb movement, examined skin histology, and analyzed gene expression to assess treatment efficacy. RESULTS: Prophylactic metformin and ASCs, whether autologous or allogeneic, effectively prevented late fibrosis, with metformin showing promising results. However, combination therapy did not provide additional benefits when used prophylactically. Autologous ASCs, alone or with metformin, proved most effective against late-stage RISF. Prophylactic intervention outperformed late therapy for mitigating radiation skin damage. Co-culture studies revealed that ASCs and metformin downregulated inflammation and fibrotic gene expression in both mouse and human fibroblasts. CONCLUSIONS: Our study suggests metformin's potential as a prophylactic measure to prevent RISF, and the combination of ASCs and metformin holds promise for late-stage RISF treatment. These findings have clinical implications for improving the quality of life for those affected by radiation-induced skin fibrosis.

Human adipose ECM alleviates radiation-induced skin fibrosis via endothelial cell-mediated M2 macrophage polarization.

Radiation therapy can lead to late radiation-induced skin fibrosis (RISF), causing movement restriction, pain, and organ dysfunction. This study evaluated adipose-derived extracellular matrix (Ad-ECM) as a mitigator of RISF. Female C57BL/6J mice that were irradiated developed fibrosis, which was mitigated by a single local Ad-ECM injection, improving limb movement and reducing epithelium thickness and collagen deposition. Ad-ECM treatment resulted in decreased expression of pro-inflammatory and fibrotic genes, and upregulation of anti-inflammatory cytokines, promoting M2 macrophage polarization. Co-culture of irradiated human fibroblasts with Ad-ECM down-modulated fibrotic gene expression and enhanced bone marrow cell migration. Ad-ECM treatment also increased interleukin (IL)-4, IL-5, and IL-15 expression in endothelial cells, stimulating M2 macrophage polarization and alleviating RISF. Prophylactic use of Ad-ECM showed effectiveness in mitigation. This study suggests Ad-ECM's potential in treating chronic-stage fibrosis.

Application of Adipose-Tissue Derived Products for Burn Wound Healing.

Burn injuries are a significant global health concern, leading to high morbidity and mortality. Deep burn injuries often result in delayed healing and scar formation, necessitating effective treatment options. Regenerative medicine, particularly cell therapy using adipose-derived stem cells (ASCs), has emerged as a promising approach to improving burn wound healing and reducing scarring. Both in vitro and preclinical studies have demonstrated the efficacy of ASCs and the stromal vascular fraction (SVF) in addressing burn wounds. The application of ASCs for burn healing has been studied in various forms, including autologous or allogeneic cells delivered in suspension or within scaffolds in animal burn models. Additionally, ASC-derived non-cellular components, such as conditioned media or exosomes have shown promise. Injection of ASCs and SVF at burn sites have been demonstrated to enhance wound healing by reducing inflammation and promoting angiogenesis, epithelialization, and granulation tissue formation through their paracrine secretome. This review discusses the applications of adipose tissue derivatives in burn injury treatment, encompassing ASC transplantation, as well as the utilization of non-cellular components utilization for therapeutic benefits. The application of ASCs in burn healing in the future will require addressing donor variability, safety, and efficacy for successful clinical application.

Nicotinamide Riboside Improves Stemness of Human Adipose-Derived Stem Cells and Inhibits Terminal Adipocyte Differentiation.

Adipose tissue plays a crucial role in maintaining metabolic homeostasis by serving as a storage site for excess fat and protecting other organs from the detrimental effects of lipotoxicity. However, the aging process is accompanied by a redistribution of fat, characterized by a decrease in insulin-sensitive subcutaneous adipose depot and an increase in insulin-resistant visceral adipose depot. This age-related alteration in adipose tissue distribution has implications for metabolic health. Adipose-derived stem cells (ASCs) play a vital role in the regeneration of adipose tissue. However, aging negatively impacts the stemness and regenerative potential of ASCs. The accumulation of oxidative stress and mitochondrial dysfunction-associated cellular damage contributes to the decline in stemness observed in aged ASCs. Nicotinamide adenine dinucleotide (NAD+) is a crucial metabolite that is involved in maintaining cellular homeostasis and stemness. The dysregulation of NAD+ levels with age has been associated with metabolic disorders and the loss of stemness. In this study, we aimed to investigate the effects of nicotinamide riboside (NR), a precursor of NAD+, on the stemness of human ASCs in cell culture. Our findings reveal that adipogenesis is accompanied by an increase in mitochondrial activity and the production of reactive oxygen species (ROS). However, treatment with NR leads to a reduction in mitochondrial activity and ROS production in ASCs. Furthermore, NR administration improves the stemness-related genes expression in ASCs and mitigates their propensity for adipocyte differentiation. These results suggest that NR treatment holds promise as a potential strategy to rejuvenate the stemness of aged ASCs. Further investigations, including in vivo evaluations using animal models and human studies, will be necessary to validate these findings and establish the clinical potential of this well-established drug for enhancing the stemness of aged stem cells.

Soluble chitosan derivative treats wound infections and promotes wound healing in a novel MRSA-infected porcine partial-thickness burn wound model.

Burns are physically debilitating and potentially fatal injuries. The most common etiology of burn wound infections in the US is methicillin-resistant Staphylococcus aureus (MRSA), which is particularly recalcitrant when biofilms form. The current standard of care, silver sulfadiazine (SSD) is effective in reducing bacterial load, but less effective in improving burn wound healing. New treatments that can manage infection while simultaneously improving healing would provide a benefit in the treatment of burns. Porcine models are frequently used as a model for human wound healing but can be expensive due to the need to separate wounds to avoid cross contamination. The porcine model developed in this study offers the capability to study multiple partial thickness burn wound (PTBW) sites on a single animal with minimal crosstalk to study wound healing, infection, and inflammation. The current study evaluates a wound rinse and a wound gel formulated with a non-toxic, polycationic chitosan derivative that is hypothesized to manage infection while also promoting healing, providing a potential alternate to SSD. Studies in vitro and in this PTBW porcine model compare treatment with the chitosan derivative formulations to SSD. The wound rinse and wound gel are observed to disrupt mature MRSA biofilms in vitro and reduce the MRSA load in vivo when compared to that of the standard of care. In vivo data further show increased re-epithelialization and faster healing in burns treated with wound rinse/gel as compared to SSD. Taken together, the data demonstrate the potential of the wound rinse/gel to significantly enhance healing, promote re-epithelialization, and reduce bacterial burden in infected PTBW using an economical porcine model.

Hydrogel patch with pretreated stem cells accelerates wound closure in diabetic rats.

Delay in wound healing is a diabetes mellites resulting disorder causing persistent microbial infections, pain, and poor quality of life. This disorder is treated by several strategies using natural biomaterials, growth factors and stem cells molded into various scaffolds which possess the potential to accelerate the closure of impaired diabetic wounds. In this study, we developed a hydrogel patch using chitosan (CS) and polyethylene glycol (PEG) with laden bone marrow-derived mesenchymal stem cells (BMSCs) that were pretreated with fibroblast growth factor 21 (FGF21). The developed hydrogel patches were characterized by scanning electron microscopy and fourier transform infrared (FTIR) spectroscopy. After studying the swelling behavior, growth factor (FGF21) was used to modulate BMSC in the hyperglycemic environment. Later, FGF21 treated BMSC were embedded in CS/PEG hydrogel patch and their wound closure effect was assessed in diabetic rats. The results showed that CS/PEG hydrogel patches have good biocompatibility and possess efficient BMSC recruiting properties. The application of CS/PEG hydrogel patches accelerated wound closure in diabetic rats as compared to the control groups. However, the use of FGF21 pretreated BMSCs laded CS/PEG hydrogel patches further increased the therapeutic efficacy of wound closure in diabetic rats. This study demonstrated that the application of a hydrogel patch of CS/PEG with FGF21 pretreated BMSCs improves diabetic wound healing, but further studies are needed on larger animals before the use of these dressings in clinical trials.

Establishment of a Robust and Reproducible Model of Radiation-Induced Skin and Muscle Fibrosis.

Radiation-induced skin fibrosis (RISF) can result from a plethora of scenarios including cancer therapy, accidental exposure, or acts of terrorism. Radioactive beams can penetrate through the skin and affect the structures in their path including skin, muscles, and internal organs. Skin is the first structure to get exposed to radiation and is susceptible to develop chronic fibrosis, which is challenging to treat. Currently, limited treatment options show moderate efficacy in mitigating radiation-related skin fibrosis. A key factor hindering the development of effective countermeasures is the absence of a convenient and robust model that could allow for translation of the experimental findings to humans. Here, a robust and reproducible murine hind limb skin fibrosis model has been established for prophylactic and therapeutic evaluation of possible agents for functional and molecular recovery. The right hind limb was irradiated using a single dose of 40 (Gray) Gy to induce skin fibrosis. Subjects developed edema and dermatitis in the early stages proceeded by visible skin constriction. Irradiated limbs showed a significantly reduced limb range of motion in the following weeks. In late stages, acute side effects subsided, yet chronic fibrosis persisted. A gait index was performed as an additional functional assay, which demonstrated the development of functional impairment. These non-invasive methods demonstrated reliable measurements for tracing fibrosis progression, which is supported by histological analyses. The radiation dose, application, and post-irradiation analyses employed in this model offer a vigorous and reproducible method for studying radiation-induced skin fibrosis and testing the efficacy of therapeutical agents.

Intestinal Radiation Protection and Mitigation by Second-Generation Probiotic Lactobacillus-reuteri Engineered to Deliver Interleukin-22.

(1) Background: The systemic administration of therapeutic agents to the intestine including cytokines, such as Interleukin-22 (IL-22), is compromised by damage to the microvasculature 24 hrs after total body irradiation (TBI). At that time, there is significant death of intestinal microvascular endothelial cells and destruction of the lamina propria, which limits drug delivery through the circulation, thus reducing the capacity of therapeutics to stabilize the numbers of Lgr5+ intestinal crypt stem cells and their progeny, and improve survival. By its direct action on intestinal stem cells and their villus regeneration capacity, IL-22 is both an ionizing irradiation protector and mitigator. (2) Methods: To improve delivery of IL-22 to the irradiated intestine, we gavaged Lactobacillus-reuteri as a platform for the second-generation probiotic Lactobacillus-reuteri-Interleukin-22 (LR-IL-22). (3) Results: There was effective radiation mitigation by gavage of LR-IL-22 at 24 h after intestinal irradiation. Multiple biomarkers of radiation damage to the intestine, immune system and bone marrow were improved by LR-IL-22 compared to the gavage of control LR or intraperitoneal injection of IL-22 protein. (4) Conclusions: Oral administration of LR-IL-22 is an effective protector and mitigator of intestinal irradiation damage.

Comparison of Clinically Relevant Adipose Preparations on Articular Chondrocyte Phenotype in a Novel In Vitro Co-Culture Model.

Adipose therapeutics, including isolated cell fractions and tissue emulsifications, have been explored for osteoarthritis (OA) treatment; however, the optimal preparation method and bioactive tissue component for healing has yet to be determined. This in vitro study compared the effects of adipose preparations on cultured knee chondrocytes. De-identified human articular chondrocytes were co-cultured with adipose preparations for 36 or 72 h. Human adipose tissues were obtained from abdominal panniculectomy procedures and processed using three different techniques: enzymatic digestion to release stromal vascular fraction (SVF), emulsification with luer-to-luer transfer (nanofat), and processing in a bead-mill (Lipogems, Lipogems International SpA, Milan, Italy). Gene expression in both chondrocytes and adipose preparations was measured to assess cellular inflammation, catabolism, and anabolism. Results demonstrated that chondrocytes cultured with SVF consistently showed increased inflammatory and catabolic gene expression compared with control chondrocytes at both 36- and 72-h timepoints. Alternatively, chondrocytes co-cultured with either nanofat or bead-mill processed adipose derivatives yielded minimal pro-inflammatory effects and instead increased anabolism and regeneration of cartilage extracellular matrix. Interestingly, nanofat preparations induced transient matrix anabolism while Lipogems adipose consistently demonstrated increased matrix synthesis at both study timepoints after co-culture. This evaluation of the regenerative potential of adipose-derived preparations as a clinical tool for knee OA treatment suggests that mechanically processed preparations may be more efficacious than an isolated SVF cell preparation.

Metformin Improves Stemness of Human Adipose-Derived Stem Cells by Downmodulation of Mechanistic Target of Rapamycin (mTOR) and Extracellular Signal-Regulated Kinase (ERK) Signaling.

Adipose tissue plays an important role in regulating metabolic homeostasis by storing excess fat and protecting other organs from lipotoxicity. Aging is associated with central fat redistribution, culminating in a decrease in insulin-sensitive subcutaneous and an increase in insulin-resistant visceral adipose depots. Adipose-derived stem cells (ASCs) play an important role in the regeneration of adipose tissue. Aged ASCs show decreased stemness and regenerative potential due to the accumulation of oxidative stress and mitochondrial dysfunction-related cell damage. Metformin is a well-established anti-diabetic drug that has shown anti-aging effects in different organisms and animal models. In this study, we analyzed the effect of metformin treatment on the stemness of human ASCs in cell culture and whole adipose tissue culture models. Our results demonstrate that metformin improves the stemness of ASCs, reducing their rate of proliferation and adipocyte differentiation. Investigating the possible underlying mechanism, we observed a decrease in the mTOR and ERK activity in metformin-treated ASCs. In addition, we observed an increase in autophagy activity upon metformin treatment. We conclude that metformin treatment improves ASCs stemness by reducing mTOR and ERK signaling and enhancing autophagy. Future in vivo evaluations in animal models and humans will pave the way for the clinical adaptation of this well-established drug for reviving the stemness of aged stem cells.

Fat Grafting in Radiation-Induced Soft-Tissue Injury: A Narrative Review of the Clinical Evidence and Implications for Future Studies.

SUMMARY: Radiation-induced changes in skin and soft tissue result in significant cosmetic and functional impairment with subsequent decrease in quality of life. Fat grafting has emerged as a therapy for radiation-induced soft-tissue injury, and this narrative review aims to evaluate the current clinical evidence regarding its efficacy. A review was conducted to examine the current clinical evidence of fat grafting as a therapy for radiation-induced injury to the skin and soft tissue and to outline the clinical outcomes that can be used to more consistently quantify chronic radiation-induced injury in future clinical studies. The current clinical evidence regarding the efficacy of fat grafting to treat radiation-induced injury of the skin and soft tissue suggests that fat grafting increases skin softness and pliability, induces volume restoration, improves hair growth in areas of alopecia, reduces pain, and improves cosmetic and functional outcomes. However, literature in this field is far from robust and mired by the retrospective nature of the studies, lack of adequate controls, and inherent limitations of small case series and cohorts. A series of actions have been identified to strengthen future clinical data, including the need for physical examination using a validated scale, appropriate imaging, skin biomechanics and microcirculation testing, and histologic analysis. In conclusion, radiation-induced soft-tissue injury is a significant health burden that can lead to severe functional and aesthetic sequelae. Although still in a preliminary research phase, there is promising clinical evidence demonstrating the benefits of fat grafting to treat chronic changes after radiation therapy. Future clinical studies will require larger cohorts, adequate controls, and consistent use of objective measurements.

Allogeneic adipose-derived stem cells mitigate acute radiation syndrome by the rescue of damaged bone marrow cells from apoptosis.

Acute radiation syndrome (ARS) is the radiation toxicity that can affect the hematopoietic, gastrointestinal, and nervous systems upon accidental radiation exposure within a short time. Currently, there are no effective and safe approaches to treat mass population exposure to ARS. Our study aimed to evaluate the therapeutic potential of allogeneic adipose-derived stem cells (ASCs) for total body irradiation (TBI)-induced ARS and understand the underlying mitigation mechanism. We employed 9.25 Gy TBI dose to C57BL/6 mice and studied the effect of allogeneic ASCs on mice survival and regeneration of the hematopoietic system. Our results indicate that intraperitoneal-injected ASCs migrated to the bone marrow, rescued hematopoiesis, and improved the survival of irradiated mice. Our transwell coculture results confirmed the migration of ASCs to irradiated bone marrow and rescue hematopoietic activity. Furthermore, contact coculture of ASCs improved the survival and hematopoiesis of irradiated bone marrow in vitro. Irradiation results in DNA damage, upregulation of inflammatory signals, and apoptosis in bone marrow cells, while coculture with ASCs reduces apoptosis via activation of DNA repair and the antioxidation system. Upon exposure to irradiated bone marrow cells, ASCs secrete prosurvival and hematopoietic factors, such as GM-CSF, MIP1α, MIP1ß, LIX, KC, 1P-10, Rantes, IL-17, MCSF, TNFα, Eotaxin, and IP-10, which reduces oxidative stress and rescues damaged bone marrow cells from apoptosis. Our findings suggest that allogeneic ASCs therapy is effective in mitigating TBI-induced ARS in mice and may be beneficial for clinical adaptation to treat TBI-induced toxicities. Further studies will help to advocate the scale-up and adaptation of allogeneic ASCs as the radiation countermeasure.

Quiescence, Stemness and Adipogenic Differentiation Capacity in Human DLK1-/CD34+/CD24+ Adipose Stem/Progenitor Cells.

We explore the status of quiescence, stemness and adipogenic differentiation capacity in adipose stem/progenitor cells (ASCs) ex vivo, immediately after isolation from human subcutaneous white adipose tissue, by sorting the stromal vascular fraction into cell-surface DLK1+/CD34-, DLK1+/CD34dim and DLK1-/CD34+ cells. We demonstrate that DLK1-/CD34+ cells, the only population exhibiting proliferative and adipogenic capacity, express ex vivo the bonafide quiescence markers p21Cip1, p27Kip1 and p57Kip2 but neither proliferation markers nor the senescence marker p16Ink4a. The pluripotency markers NANOG, SOX2 and OCT4 are barely detectable in ex vivo ASCs while the somatic stemness factors, c-MYC and KLF4 and the early adipogenic factor C/EBPß are highly expressed. Further sorting of ASCs into DLK1-/CD34+/CD24- and DLK1-/CD34+/CD24+ fractions shows that KLF4 and c-MYC are higher expressed in DLK1-/CD34+/CD24+ cells correlating with higher colony formation capacity and considerably lower adipogenic activity. Proliferation capacity is similar in both populations. Next, we show that ASCs routinely isolated by plastic-adherence are DLK1-/CD34+/CD24+. Intriguingly, CD24 knock-down in these cells reduces proliferation and adipogenesis. In conclusion, DLK1-/CD34+ ASCs in human sWAT exist in a quiescent state, express high levels of somatic stemness factors and the early adipogenic transcription factor C/EBPß but senescence and pluripotency markers are barely detectable. Moreover, our data indicate that CD24 is necessary for adequate ASC proliferation and adipogenesis and that stemness is higher and adipogenic capacity lower in DLK1-/CD34+/CD24+ relative to DLK1-/CD34+/CD24- subpopulations.

The Impact of Human Lipoaspirate and Adipose Tissue-Derived Stem Cells Contact Culture on Breast Cancer Cells: Implications in Breast Reconstruction.

BACKGROUND: Autologous fat transfer in the form of lipoaspirates for the reconstruction of the breast after breast cancer surgery is a commonly used procedure in plastic surgery. However, concerns regarding the oncologic risk of nutrient-rich fat tissue are widely debated. Previous studies have primarily focused on studying the interaction between adipose-derived stem cells (ASCs) and breast cancer cells. METHODS: In this study, we performed a comprehensive analysis of the paracrine- and contact-based interactions between lipoaspirates, ASCs and breast cancer cell lines. An inverted flask culture method was used to study the contact-based interaction between lipoaspirates and breast cancer cells, while GFP-expressing breast cancer cell lines were generated to study the cell-cell contact interaction with ASCs. Three different human breast cancer cell lines, MCF-7, MDA-MB-231 and BT-474, were studied. We analyzed the impact of these interactions on the proliferation, cell cycle and epithelial-to-mesenchymal (EMT) transition of the breast cancer cells. RESULTS: Our results revealed that both lipoaspirates and ASCs do not increase the proliferation rate of the breast cancer cells either through paracrine- or contact-dependent interactions. We observed that lipoaspirates selectively inhibit the proliferation of MCF-7 cells in contact co-culture, driven by the retinoblastoma (Rb) protein activity mediating cell cycle arrest. Additionally, ASCs inhibited MDA-MB-231 breast cancer cell proliferation in cell-cell contact-dependent interactions. Quantitative real-time PCR revealed no significant increase in the EMT-related genes in breast cancer cells upon co-culture with ASCs. CONCLUSION: In conclusion, this study provides evidence of the non-oncogenic character of lipoaspirates and supports the safety of clinical fat grafting in breast reconstruction after oncological surgical procedures. In vivo studies in appropriate animal models and long-term post-operative clinical data from patients are essential to reach the final safety recommendations.

CRISPR/Cas9-mediated gene knockout in human adipose stem/progenitor cells.

The CRISPR/Cas9 system is a powerful tool to generate a specific loss-of-function phenotype by gene knockout (KO). However, this approach is challenging in primary human cells. In this technical report, we present a reliable protocol to achieve a functional KO in the genome of human adipose stem/progenitor cells (ASCs). Using Sprouty1 (SPRY1) as a model target gene for a CRISPR/Cas9 mediated KO, we particularize the procedure including the selection of the CRISPR/Cas9 target sequences and the employment of appropriate lentiviral vectors to obtain a functional gene KO. The efficiency of CRISPR/Cas9 to mutate the SPRY1 gene is determined by a PCR-based mutation detection assay and sequence analysis. Effects on mRNA and protein levels are studied by RT-qPCR and Western blotting. In addition, we demonstrate that CRISPR/Cas9 mediated SPRY1 KO and gene silencing by shRNA are similarly effective to deplete the Sprouty1 protein and to inhibit adipogenic differentiation. In summary, we show a reliable approach to achieve a gene KO in human ASCs, which could also apply to other primary cell types. Abbreviations: ASC: Adipogenic Stem/Progenitor Cell; Cas: CRISPR-associated system; CRISPR: Clustered Regularly Interspaced Palindromic Repeat; gDNA: Genomic DNA; GOI: Gene of interest; gRNA: Guide RNA; NHEJ: Non-homologous end joining; Indel: Insertion/Deletion; PAM: Protospacer adjacent motif; sWAT: Subcutaneous white adipose tissue; TIDE: Tracking of indels by decomposition.

Curcumin preconditioned human adipose derived stem cells co-transplanted with platelet rich plasma improve wound healing in diabetic rats.

AIM: Inflammatory and oxidative microenvironment at diabetic' wound site hinder the therapeutic efficacy of cell-based therapies in diabetic patients. The purpose of this study is to explore the competence of curcumin preconditioned human adipose derived cells (hASCs) in combination with platelet rich plasma (PRP) for the repair of wounds in diabetic rats. MAIN METHODS: The cytoprotective effect of curcumin preconditioning for hASCs against hyperglycemic stress was evaluated through analysis of cell morphology, viability, cytotoxicity, senescence, and scratch wound healing assays. Subsequently, the healing capacity of curcumin preconditioned hASCs (Cur-hASCs) added to PRP was examined in excisional wounded diabetic rat model. Healed skin biopsies were excised to analyze gene and protein expression of wound healing markers by qPCR and western blotting. Histopathological changes were observed through hematoxylin and eosin staining. KEY FINDINGS: We found that Cur-hASCs counteract the glucose stress much better than non-preconditioned hASCs by maintaining their cellular morphology and viability as well as metabolic potential. Further in vivo results revealed that, Cur-hASCs co-injected with PRP resulted in faster wound closure, improved fibroblast proliferation, increased neovascularization, marked reduction in inflammatory cells, and compact extracellular matrix with completely covered thick epithelium. Moreover, Cur-hASCs + PRP treatment significantly improved the expression of key healing markers such as pro-angiogenic (Vegf), dermal matrix deposition (Col1α1), cell migration (bFgf) and cell proliferation (Pcna) at wound site. SIGNIFICANCE: Our findings propose a combinatorial therapy (Cur-hASCs + PRP) as a novel modality to improve the efficacy of hASCs-based therapy for diabetic wounds.

Sprouty1 Prevents Cellular Senescence Maintaining Proliferation and Differentiation Capacity of Human Adipose Stem/Progenitor Cells.

The role of Ras-Mitogen-activated protein kinase (MAPK) signaling in cellular aging is not precisely understood. Recently, we identified Sprouty1 (SPRY1) as a weight-loss target gene in human adipose stem/progenitor cells (ASCs) and showed that Sprouty1 is important for proper regulation of adipogenesis. In the present study, we show that loss-of-function of Sprouty1 by CRISPR/Cas9-mediated genome editing in human ASCs leads to hyper-activation of MAPK signaling and a senescence phenotype. Sprouty1 knockout ASCs undergo an irreversible cell cycle arrest, become enlarged and stain positive for senescence-associated ß-galactosidase. Sprouty1 down-regulation leads to DNA double strand breaks, a considerably increased number of senescence-associated heterochromatin foci and induction of p53 and p21Cip1. In addition, we detect an increase of hypo-phosphorylated Retinoblastoma (Rb) protein in SPRY1 knockout ASCs. p16Ink4A is not induced. Moreover, we show that Sprouty1 knockout leads to induction of a senescence-associated secretory phenotype as indicated by the activation of the transcription factors NFκB and C/EBPß and a significant increase in mRNA expression and secretion of interleukin-8 (IL-8) and CXCL1/GROα. Finally, we demonstrate that adipogenesis is abrogated in senescent SPRY1 knockout ASCs. In conclusion, this study reveals a novel mechanism showing the importance of Sprouty1 for the prevention of senescence and the maintenance of the proliferation and differentiation capacity of human ASCs.

Epigallocatechin-3-gallate protects Wharton's jelly derived mesenchymal stem cells against in vitro heat stress.

The deteriorating effects of heat stress abrogate the therapeutic implications of human Wharton's jelly derived mesenchymal stem cells (hWJMSCs) transplanted in burn wounds. Topically applied green tea extract comprising epigallocatechin-3-gallate (EGCG) is known to repair burn wounds. Here, we investigated the protective role of EGCG priming of hWJMSCs against heat-induced stress in vitro along with the involved underlying mechanism. EGCG ameliorated heat-induced injuries as demonstrated by significantly improved cell morphology, viability, triggered cell migration and enhanced expression of heat shock proteins. In addition, decreased lactate dehydrogenase release and reduced percentage of senescent and apoptotic cells were observed. EGCG priming alleviated the detrimental effects of thermal stress in hWJMSCs as observed by significant down-regulation in expression of BCL2 associated X (BAX), interleukin 6 (IL6), and interleukin 1 beta (IL1ß) genes, while proliferating cell nuclear antigen (PCNA), BCL2 like 1 (BCL2L1), vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGFß1), hepatocyte growth factor (HGF) and interleukin 4 (IL4) genes were up-regulated. Accompanying gene expression data, EGCG primed cells exposed to heat stress also exhibited remarkably increased secretion of VEGF, HGF, epidermal growth factor (EGF), stromal-derived factor 1 (SDF1) proteins while the reduced release of IL-6, and tumor necrosis factor-alpha (TNF-α) proteins. Further, synergistic activation of extracellular-signal-regulated kinase (ERK) and protein kinase B (PKB/AKT) proteins was observed. These findings suggest that EGCG priming might enhance the therapeutic efficacy of hWJMSCs in the burnt tissue through regulation of ERK and AKT signaling pathways, and improved cellular responses.