establishment-robust-reproducible-model-radiation-induced-skin-muscle

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. ...

University of Pittsburgh

Radiation therapy to anatomic regions, including the head and neck, chest wall, and extremities, can produce radiation-induced fibrosis (RIF). To elucidate the cellular and molecular mechanism(s) involved in RIF, female C57BL/6J mice were irradiated to the right flank to 35 Gy in single fraction using 6 Mv electrons. Radiation fibrosis was detected by day 14, was increased by day 28, and confirmed by Masson's trichrome histological staining for collagen. Biopsied tissue at day 14 showed an increase in expression of fibrosis-related genes including transforming growth factor-β (TGF-β) and collagens 1-6. A single adipose-derived stem cell (ASC) injection on day 28 at the irradiated site decreased by day 40: epithelial thickness, collagen deposition, and significantly improved limb excursion compared with irradiated controls. Noncontact transwell coculture of ASCs above a monolayer of irradiated human foreskin fibroblasts downregulated fibrosis-related genes TGF-β, connective tissue growth factor, interleukin-1, NF-kB, tumor necrosis factor, and collagens 1-6. Hepatocyte growth factor (HGF) secreted by ASCs was identified as a novel mechanism by which ASCs exert antifibrotic effects by downregulating fibrotic gene expression in irradiated cells and recruiting bone marrow cells to the irradiated site. In conclusion, these data indicate a mechanistic role of HGF secreted by ASCs in reducing RIF. Stem Cells 2019;37:791-802.

Adipose-Derived Stem Cell Therapy Ameliorates Ionizing Irradiation Fibrosis via Hepatocyte Growth Factor-Mediated Transforming Growth Factor-β Downregulation and Recruitment of Bone Marrow Cells.

The bone marrow (BM) is a major reservoir of resting memory T cells and long-lived plasma cells, capable of providing protection against recurrent infections. Whether the age-related accumulation of adipose tissue in the BM affects the functionality and maintenance of memory cells is not well understood.

Human bone marrow adipocytes display distinct immune regulatory properties.

Radiation therapy has been increasingly employed as a tool to cure and palliate majority of solid tumors. Although radiotherapy has shown promising results in preserving structure and function of organs, it is associated with late side effects mainly manifested in the form of tissue fibrosis. Recent advances in molecular biology techniques has helped better understand the molecular mechanisms involved in radiation induced fibrosis. Currently, very few treatment modalities are available to treat the condition with moderate success rate. Stem cell therapies and particularly adipose tissue and adipose derived stem cells therapies have shown promising results in clinical applications. Identification of the key factors involved in the mitigation process will help to enhance the beneficial effects and develop new therapy approaches.

Understanding the mechanism of radiation induced fibrosis and therapy options.

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 (IL1β) genes, while proliferating cell nuclear antigen (PCNA), BCL2 like 1 (BCL2L1), vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGFβ1), 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.

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

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.

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

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 () 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  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  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.  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: sertion/etion; PAM: Protospacer adjacent motif; sWAT: Subcutaneous white adipose tissue; TIDE: Tracking of indels by decomposition.

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

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.

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

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/CD34 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 p21, p27 and p57 but neither proliferation markers nor the senescence marker p16. 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/EBPβ 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/EBPβ 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.

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

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, MIP1β, 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.

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

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.

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

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.

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 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.

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

(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  as a platform for the second-generation probiotic -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.

Article	Year
Adipose-Derived Stem Cell Therapy Ameliorates Ionizing Irradiation Fibrosis via Hepatocyte Growth Factor-Mediated Transforming Growth Factor-β Downregulation and Recruitment of Bone Marrow C... Stem cells (Dayton, Ohio)\| PubMed ID: 30861238	2019
Human bone marrow adipocytes display distinct immune regulatory properties. EBioMedicine\| PubMed ID: 31327694	2019
Understanding the mechanism of radiation induced fibrosis and therapy options. Pharmacology & therapeutics\| PubMed ID: 31419518	2019
Epigallocatechin-3-gallate protects Wharton's jelly derived mesenchymal stem cells against in vitro heat stress. European journal of pharmacology\| PubMed ID: 32001222	2020
Curcumin preconditioned human adipose derived stem cells co-transplanted with platelet rich plasma improve wound healing in diabetic rats. Life sciences\| PubMed ID: 32668325	2020
CRISPR/Cas9-mediated gene knockout in human adipose stem/progenitor cells. Adipocyte\| PubMed ID: 33070670	2020
The Impact of Human Lipoaspirate and Adipose Tissue-Derived Stem Cells Contact Culture on Breast Cancer Cells: Implications in Breast Reconstruction. International journal of molecular sciences\| PubMed ID: 33271950	2020
Quiescence, Stemness and Adipogenic Differentiation Capacity in Human DLK1/CD34/CD24 Adipose Stem/Progenitor Cells. Cells\| PubMed ID: 33498986	2021
Allogeneic adipose-derived stem cells mitigate acute radiation syndrome by the rescue of damaged bone marrow cells from apoptosis. Stem cells translational medicine\| PubMed ID: 33724714	2021
Fat Grafting in Radiation-Induced Soft-Tissue Injury: A Narrative Review of the Clinical Evidence and Implications for Future Studies. Plastic and reconstructive surgery\| PubMed ID: 33776031	2021
Metformin Improves Stemness of Human Adipose-Derived Stem Cells by Downmodulation of Mechanistic Target of Rapamycin (mTOR) and Extracellular Signal-Regulated Kinase (ERK) Signaling. Biomedicines\| PubMed ID: 34944598	2021
Comparison of Clinically Relevant Adipose Preparations on Articular Chondrocyte Phenotype in a Novel In Vitro Co-Culture Model. Stem cells and development\| PubMed ID: 35442089	2022
Intestinal Radiation Protection and Mitigation by Second-Generation Probiotic Engineered to Deliver Interleukin-22. International journal of molecular sciences\| PubMed ID: 35628427	2022

Asim Ejaz

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