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Differentiation of diabetic foot ulcer-derived induced pluripotent stem cells reveals distinct cellular and tissue phenotypes.

ABSTRACT: Diabetic foot ulcers (DFUs) are a major complication of diabetes, and there is a critical need to develop novel cell- and tissue-based therapies to treat these chronic wounds. Induced pluripotent stem cells (iPSCs) offer a replenishing source of allogeneic and autologous cell types that may be beneficial to improve DFU wound-healing outcomes. However, the biologic potential of iPSC-derived cells to treat DFUs has not, to our knowledge, been investigated. Toward that goal, we have performed detailed characterization of iPSC-derived fibroblasts from both diabetic and nondiabetic patients. Significantly, gene array and functional analyses reveal that iPSC-derived fibroblasts from both patients with and those without diabetes are more similar to each other than were the primary cells from which they were derived. iPSC-derived fibroblasts showed improved migratory properties in 2-dimensional culture. iPSC-derived fibroblasts from DFUs displayed a unique biochemical composition and morphology when grown as 3-dimensional (3D), self-assembled extracellular matrix tissues, which were distinct from tissues fabricated using the parental DFU fibroblasts from which they were reprogrammed. In vivo transplantation of 3D tissues with iPSC-derived fibroblasts showed they persisted in the wound and facilitated diabetic wound closure compared with primary DFU fibroblasts. Taken together, our findings support the potential application of these iPSC-derived fibroblasts and 3D tissues to improve wound healing.-Kashpur, O., Smith, A., Gerami-Naini, B., Maione, A. G., Calabrese, R., Tellechea, A., Theocharidis, G., Liang, L., Pastar, I., Tomic-Canic, M., Mooney, D., Veves, A., Garlick, J. A. Differentiation of diabetic foot ulcer-derived induced pluripotent stem cells reveals distinct cellular and tissue phenotypes.

SUBMITTER: Kashpur O

PROVIDER: S-EPMC6355091 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Differentiation of diabetic foot ulcer-derived induced pluripotent stem cells reveals distinct cellular and tissue phenotypes.

Kashpur Olga O Smith Avi A Gerami-Naini Behzad B Maione Anna G AG Calabrese Rossella R Tellechea Ana A Theocharidis Georgios G Liang Liang L Pastar Irena I Tomic-Canic Marjana M Mooney David D Veves Aristidis A Garlick Jonathan A JA

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20180808 1

Diabetic foot ulcers (DFUs) are a major complication of diabetes, and there is a critical need to develop novel cell- and tissue-based therapies to treat these chronic wounds. Induced pluripotent stem cells (iPSCs) offer a replenishing source of allogeneic and autologous cell types that may be beneficial to improve DFU wound-healing outcomes. However, the biologic potential of iPSC-derived cells to treat DFUs has not, to our knowledge, been investigated. Toward that goal, we have performed detai ...[more]

PMID: 30088952

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Project description:PurposeWe aimed to investigate the prevalence of diabetic retinopathy (DR) in patients with diabetic foot ulcer (DFU) and elucidate the association between DR and DFU severities and their shared risk factors.MethodsA retrospective review was conducted on DFU patients who underwent ophthalmic and vascular examinations within 6 months; 100 type 2 diabetic patients with DFU were included. The medical records of 2496 type 2 diabetic patients without DFU served as control data. DR prevalence and severity were assessed in DFU patients. DFU patients were compared with the control group regarding each clinical variable. Additionally, DFU patients were divided into two groups according to DR severity and compared.ResultsOut of 100 DFU patients, 90 patients (90%) had DR and 55 (55%) had proliferative DR (PDR). There was no significant association between DR and DFU severities (R = 0.034, p = 0.734). A multivariable analysis comparing type 2 diabetic patients with and without DFUs showed that the presence of DR [OR, 226.12; 95% confidence interval (CI), 58.07-880.49; p < 0.001] and proliferative DR [OR, 306.27; 95% CI, 64.35-1457.80; p < 0.001), higher HbA1c (%, OR, 1.97, 95% CI, 1.46-2.67; p < 0.001), higher serum creatinine (mg/dL, OR, 1.62, 95% CI, 1.06-2.50; p = 0.027), older age (years, OR, 1.12; 95% CI, 1.06-1.17; p < 0.001), higher pulse pressure (mmHg, OR, 1.03; 95% CI, 1.00-1.06; p = 0.025), lower cholesterol (mg/dL, OR, 0.94; 95% CI, 0.92-0.97; p < 0.001), lower BMI (kg/m2, OR, 0.87, 95% CI, 0.75-1.00; p = 0.044) and lower hematocrit (%, OR, 0.80, 95% CI, 0.74-0.87; p < 0.001) were associated with DFUs. In a subgroup analysis of DFU patients, the PDR group had a longer duration of diabetes mellitus, higher serum BUN, and higher serum creatinine than the non-PDR group. In the multivariable analysis, only higher serum creatinine was associated with PDR in DFU patients (OR, 1.37; 95% CI, 1.05-1.78; p = 0.021).ConclusionsDiabetic retinopathy is prevalent in patients with DFU and about half of DFU patients had PDR. No significant association was found in terms of the severity of these two diabetic complications. To prevent blindness, patients with DFU, and especially those with high serum creatinine, should undergo retinal examinations for timely PDR diagnosis and management.

Project description:Accurate identification of pathogens, rather than colonising bacteria, is a prerequisite for targeted antibiotic therapy to ensure optimal patient outcome in wounds, such as diabetic foot ulcers. Wound swabs are the easiest and most commonly used sampling technique but most published guidelines recommend instead removal of a tissue sample from the wound bed, which is a more complex process. The aim of this study was to assess the concordance between culture results from wound swabs and tissue samples in patients with suspected diabetic foot infection. Patients with a diabetic foot ulcer that is thought to be infected are being recruited from 25 sites across England in a cross-sectional study. The coprimary endpoints for the study are agreement between the two sampling techniques for three microbiological parameters: reported presence of likely isolates identified by the UK Health Protection Agency; resistance of isolates to usual antibiotic agents; and, the number of isolates reported per specimen. Secondary endpoints include appropriateness of the empiric antibiotic therapy prescribed and adverse events. Enrolling 400 patients will provide 80% power to detect a difference of 3% in the reported presence of an organism, assuming organism prevalence of 10%, discordance of 5% and a two-sided test at the 5% level of significance. Assumed overall prevalence is based on relatively uncommon organisms such as Pseudomonas. We will define acceptable agreement as κ>0.6. Concordance in diabetic foot ulcer infection (CODIFI) will produce robust data to evaluate the two most commonly used sampling techniques employed for patients with a diabetic foot infection. This will help determine whether or not it is important that clinicians take tissue samples rather than swabs in infected ulcers. This study has been approved by the Sheffield NRES Committee (Ref: 11/YH/0078) and all sites have obtained local approvals prior starting recruitment. NRES Ref: 11/YH/0078, UKCRN ID: 10440, ISRCTN: 52608451.

Project description:OBJECTIVE:To determine the extent of agreement and patterns of disagreement between wound swab and tissue samples in patients with an infected diabetic foot ulcer (DFU). DESIGN:Multicentre, prospective, cross-sectional study. SETTING:Primary and secondary care foot ulcer/diabetic outpatient clinics and hospital wards across England. PARTICIPANTS:Inclusion criteria: consenting patients aged ?18 years; diabetes mellitus; suspected infected DFU. EXCLUSION CRITERIA:clinically inappropriate to take either sample. INTERVENTIONS:Wound swab obtained using Levine's technique; tissue samples collected using a sterile dermal curette or scalpel. OUTCOME MEASURES:Coprimary: reported presence, and number, of pathogens per sample; prevalence of resistance to antimicrobials among likely pathogens. Secondary: recommended change in antibiotic therapy based on blinded clinical review; adverse events; sampling costs. RESULTS:400 consenting patients (79% male) from 25 centres.Most prevalent reported pathogens were Staphylococcus aureus (43.8%), Streptococcus (16.7%) and other aerobic Gram-positive cocci (70.6%). At least one potential pathogen was reported from 70.1% of wound swab and 86.1% of tissue samples. Pathogen results differed between sampling methods in 58% of patients, with more pathogens and fewer contaminants reported from tissue specimens.The majority of pathogens were reported significantly more frequently in tissue than wound swab samples (P<0.01), with equal disagreement for S. aureus and Pseudomonas aeruginosa. Blinded clinicians more often recommended a change in antibiotic regimen based on tissue compared with wound swab results (increase of 8.9%, 95%?CI 2.65% to 15.3%). Ulcer pain and bleeding occurred more often after tissue collection versus wound swabs (pain: 9.3%, 1.3%; bleeding: 6.8%, 1.5%, respectively). CONCLUSION:Reports of tissue samples more frequently identified pathogens, and less frequently identified non-pathogens compared with wound swab samples. Blinded clinicians more often recommended changes in antibiotic therapy based on tissue compared with wound swab specimens. Further research is needed to determine the effect of the additional information provided by tissue samples. TRIAL REGISTRATION NUMBER:ISRCTN52608451.

Project description:BackgroundDiabetic foot ulcers (DFU) are characterised by high levels of inflammatory mediators, resulting from sustained hyperglycaemic insult and the local microbial biofilm. The intralesional administration of epidermal growth factor (EGF) has emerged as an effective treatment that stimulates granulation and closure of DFU, reducing the risk of amputation. Within the wound, fibroblasts play key roles during the healing process, promoting granulation and contraction. The aim of the present study was to examine the anti-inflammatory effect of EGF in DFU-derived fibroblasts, challenged with lipopolysaccharide (LPS), under hyperglycaemic conditions, recreating in vitro what happens in a clinical scenario.MethodsHealthy skin (HS) and DFU granulation tissue biopsies were used to isolate primary fibroblasts. The effect of LPS on cell proliferation was analysed. Transcriptional expression of toll-like receptor (TLR) pathway mediators (TLR4, TLR2, CD14, MYD88 and NFKB) and pro-inflammatory cytokines (TNF, IL-6 and IL-1B) were measured by semi-quantitative polymerase chain reaction (qPCR), in cells treated with appropriate concentrations of LPS, EGF and their combination. IL-6 protein concentration was quantified by ELISA.ResultsLPS stimulated proliferation of HS-derived fibroblasts, while inhibiting the proliferation of cells derived from DFU at the highest assayed concentration of 1 µg/mL. Regarding the TLR signalling pathway, LPS increased messenger RNA levels of mediators and pro-inflammatory genes, while EGF, alone or in the presence of LPS, downregulated them, except for IL-1B.ConclusionThe results suggest that EGF might elicit an anti-inflammatory response in LPS-challenged fibroblasts, even in a hyperglycaemic milieu. Collectively, our findings contribute to explain newly observed effects of EGF in the clinical arena.Lay summaryIn this research article, we analyse the putative anti-inflammatory effect of epidermal growth factor (EGF) on fibroblast isolated from diabetic foot ulcer (DFU) granulation tissue. To induce the inflammatory response, the cells were treated with lipopolysaccharide (LPS), simulating the gram-negative bacterial infection that takes place in the wounds of diabetic patients. We studied the expression of genes involved in bacterial recognition receptors signalling pathway and those that code for different pro-inflammatory cytokines.We obtained primary fibroblasts from biopsies of a neuropathic diabetic ulcer and from healthy skin, the former was used as the control. Cells were isolated and grown in high glucose Dulbecco's Modified Eagle Medium (DMEM) culture medium, to simulate the hyperglycaemic insult. The effect of increasing concentrations of LPS on cell proliferation was analysed. Relative transcriptional expression of genes in the study was quantified by quantitative polymerase chain reaction (qPCR) in cells treated with LPS, EGF or a combination. Untreated cells served to normalise the expression.In the present study, we demonstrated that EGF modulated the primary immune response by reducing the activation of pathogen-recognition receptors and common genes involved in these signalling pathways, even in hyperglycaemic conditions. This effect translated in a decreased expression of pro-inflammatory cytokines. These results contribute to explain our previous observations about the reduction of circulating levels of inflammatory cytokines after local administration of human recombinant EGF in DFU. Further molecular studies should be carried out to fully understand the biological mechanisms elicited by EGF in this clinical scenario.

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Differentiation of diabetic foot ulcer-derived induced pluripotent stem cells reveals distinct cellular and tissue phenotypes.

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Differentiation of diabetic foot ulcer-derived induced pluripotent stem cells reveals distinct cellular and tissue phenotypes.

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