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Platelet-Rich Plasma (PRP) is an autologous biological therapy obtained by centrifuging the patient’s own blood to concentrate platelets. The addition of autologous thrombin and calcium chloride to PRP allows the production of a semi-solid form called PRP gel. PRP gel is increasingly used in a variety of tissue defects and predominantly in the management of non-healing chronic wounds. The topical application of PRP gel seems promising due to the capability of platelets to store and secrete growth factors (GF), fibrin and cytokines, which are essentials for wound healing. Most patients who suffered from chronic wounds are elderly patients with co-morbidities and polypharmacy including antithrombotic drugs such as antiplatelet agents (AP) or anticoagulants (AC), which could hamper the feasibility of this autologous platelet-derived therapy. To date, no study has investigated PRP gel formation in patients with AP or AC. The aim of this study was to evaluate the influence of AP or AC drugs on the production of PRP gel formation from elderly patients. Different biological characteristics were determined to qualify the production of PRP gel from such patients (Interquartile range (IQR) = 75-92 years) compared to healthy volunteers (IQR = 23-37 years). No significant difference was observed in the volume, composition (quantity of platelets, leukocytes and red blood cells) and functionality of platelets from PRP except a higher ADP-induced P-selectin expression in healthy donors compared with elderly patients. Autologous thrombin characteristics were similar in the two groups. Gel time formation (IQR: 120-195 seconds for controls and 135-210 seconds for elderly patients) and final composition of PRP gel were not significantly modified. Concentrations of theoretical thrombin generated in the serum and in the gel were inversely correlated with the time of formation of PRP gel (r2 = 0.57, p = 0.012). Altogether these data indicate that PRP gel preparation is not impacted by the use of antithrombotic drugs. Such results support the feasibility of using this innovative autologous biotherapy in the management of elderly patients with non-healing chronic wounds. [ABSTRACT FROM AUTHOR]

Adipose tissue is recognized as a valuable source of cells with angiogenic, immunomodulatory, reparative and antifibrotic properties and emerged as a therapeutic alternative for the regeneration and repair of damaged tissues. The use of adipose-tissue-based therapy is expanding in autoimmune diseases, particularly in Systemic Sclerosis (SSc), a disease in which hands and face are severely affected, leading to disability and a decrease in quality of life. Combining the advantage of an abundant supply of fat tissue and a high abundance of stem/stromal cells, fat grafting and adipose tissue-derived cell-based therapies are attractive therapeutic options in SSc. This review aims to synthesize the evidence to determine the effects of the use of these biological products for face and hands treatment in the context of SSc. This highlights several points: the need to use relevant effectiveness criteria taking into account the clinical heterogeneity of SSc in order to facilitate assessment and comparison of innovative therapies; second, it reveals some impacts of the disease on fat-grafting success; third, an important heterogeneity was noticed regarding the manufacturing of the adipose-derived products and lastly, it shows a lack of robust evidence from controlled trials comparing adipose-derived products with standard care.

Objectives/hypothesis: Vocal folds (VF) scarring leads to severe dysphonia which negatively impacts daily life of patients. Current therapeutic options are limited due in large part to the high complexity of the micro-structure of the VF. Innovative therapies derived from adipose tissue such as stromal vascular fraction (SVF) or adipose derived stromal/ stem cells (ASC) are currently being evaluated in this indication and paracrine anti-fibrotic effects are considered as predominant mechanisms., Methods: The paracrine anti-fibrotic effects of SVF and ASC from healthy donors were tested in an innovative in vitro fibrogenesis model employing human VF fiboblasts (hVFF) and the principles of macromolecular crowding (MMC). Biosynthesis of collogen and alpha-smooth-muscle actin (αSMA) expression in hVFF were quantified after five days of indirect coculture with ASC or SVF using silver stain, western blot and RT-qPCR analysis., Results: Fibrogenesis was promoted by addition of transforming growth factor beta 1 (TGFβ1) combined with MMC characterized by an enhanced deposition of fibrillar collagens and the acquisition of a myofibroblast phenotype (overexpression of αSMA). Adipose-derived therapies led to a reduction in the αSMA expression and the collagen content was lower in hVFF co-cultivated with SVF., Conclusions: ASC and SVF promoted significant prevention of fibrosis in an in vitro fibrogenesis model through paracrine mechanisms, supporting further development of adipose-derived cellular therapies in VF scarring., (Copyright © 2022 The Voice Foundation. Published by Elsevier Inc. All rights reserved.)

Systemic sclerosis (SSc) is a complex autoimmune disease characterized by a functional and structural alteration of the microvascular network associated with cutaneous and visceral fibrosis lesions. Conventional therapies are based on the use of immunomodulatory molecules and symptomatic management but often prove to be insufficient, particularly for patients suffering from severe and rapidly progressive forms of the disease. In this context, cellular therapy approaches could represent a credible solution with the goal to act on the different components of the disease: the immune system, the vascular system and the extracellular matrix. The purpose of this review is to provide an overview of the cellular therapies available for the management of SSc. The first part will focus on systemically injected therapies, whose primary effect is based on immunomodulatory properties and immune system resetting, including autologous hematopoietic stem cell transplantation and intravenous injection of mesenchymal stem cells. The second part will discuss locally administered regenerative cell therapies, mainly derived from adipose tissue, developed for the management of local complications as hand and face disabilities., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Coronavirus disease 2019 (COVID-19) has become one of the biggest public health challenges of this century. Severe forms of the disease are associated with a thrombo-inflammatory state that can turn into thrombosis. Because tissue factor (TF) conveyed by extracellular vesicles (EVs) has been implicated in thrombosis, we quantified the EV-TF activity in a cohort of hospitalized patients with COVID-19 (n = 111) and evaluated its link with inflammation, disease severity, and thrombotic events. Patients with severe disease were compared with those who had moderate disease and with patients who had septic shock not related to COVID-19 (n = 218). The EV-TF activity was notably increased in patients with severe COVID-19 compared with that observed in patients with moderate COVID-19 (median, 231 [25th to 75th percentile, 39-761] vs median, 25 [25th to 75th percentile, 12-59] fM; P < .0001); EV-TF was correlated with leukocytes, D-dimer, and inflammation parameters. High EV-TF values were associated with an increased thrombotic risk in multivariable models. Compared with patients who had septic shock, those with COVID-19 were characterized by a distinct coagulopathy profile with significantly higher EV-TF and EV-fibrinolytic activities that were not counterbalanced by an increase in plasminogen activator inhibitor-1 (PAI-1). Thus, this article is the first to describe the dissemination of extreme levels of EV-TF in patients with severe COVID-19, which supports the international recommendations of systematic preventive anticoagulation in hospitalized patients and potential intensification of anticoagulation in patients with severe disease., (© 2021 by The American Society of Hematology.)

Beside the commonly described pulmonary expression of the coronavirus disease 2019 (COVID-19), major vascular events have been reported. The objective of this study was to investigate whether increased levels of circulating endothelial cells (CECs) might be associated with severe forms of COVID-19. Ninety-nine patients with COVID-19 were enrolled in this retrospective study. Patients in the intensive care units (ICU) had significantly higher CEC counts than non-ICU patients and the extent of endothelial injury was correlated with putative markers of disease severity and inflammatory cytokines. Together, these data provide in vivo evidence that endothelial injury is a key feature of COVID-19., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: External radiotherapy has become indispensable in oncological therapies. Unfortunately, radiation is responsible for serious side effects, such as radiodermatitis. The skin is weakened and ulcerated. Our study aimed to evaluate the subcutaneous transfer of microfat (MF) alone and two mixes: MF+Platelet-rich plasma (PRP) and MF+stromal vascular fraction (SVF) to treat radiation-induced skin lesions., Method: We defined randomly five experimental groups of nine mice: 1 healthy control group and 4 irradiated (60 Grey) and treated groups. The skin lesions were treated 3 months after irradiation by MF, MF+PRP (50%-50%), MF+SVF (90%-10%) or Ringer-lactate subcutaneous injections. Wound healing was evaluated at 1, 2 and 3 months post-injection and histological wound analysis at 3 months, after euthanasia., Results: All the irradiated mice presented with wounds. After sham-injection, the wound area increased by 91.1±71.1% versus a decrease of 15.9±23.1% after MF alone (NS), 27.3±23.8% after MF+SVF (NS) and 76.4±7.7% after MF+PRP (P=0.032). A significative reduction of skin thickness in wound periphery was measured for the three treated groups compared to sham-injection (P<0.05) but not in the healed wounds (NS). The most important subcutaneous neo-vessel density was shown after MF+SVF injection., Conclusion: The MF+PRP mix was the most efficient product to increase healing. The MF+SVF mix showed the highest rate of neo-angiogenesis but was disappointing in terms of healing., Level of Evidence: Not gradable., (Copyright © 2020 Elsevier Ltd and ISBI. All rights reserved.)

The therapeutic use of adipose-derived stromal vascular fraction (SVF) is expanding in multiple pathologies. Various processes have been proposed for manufacturing SVF but they must be revisited based on advanced therapy medicinal product (ATMP) regulations. We report here the development and validation of a fully good manufacturing practices (GMP)-compliant protocol for the isolation of SVF. Adipose tissue was collected from healthy volunteers undergoing lipoaspiration. The optimal conditions of collagenase digestion and washing were determined based on measurements of SVF cell viability, yield recovery, and cell subset distribution. Comparability of the SVF obtained using the newly developed manufacturing process (n = 6) and the Celution-based automated method (n = 33), used as a reference, was established using inter-donor analyses. Characteristics of SVF (n = 5) generated using both manufacturing protocols were analyzed for an intra-donor comparison. In addition, these comparisons also included the determination of colony-forming unit fibroblast frequency, in vitro angiogenic activity, and in vivo regenerative effects in a mouse ischemic cutaneous wound model. We successfully developed a process for the generation of SVF presenting higher cell viability and yield recovery compared to the Celution device-based protocol. Characteristics of the SVF including phenotype, capacity for angiogenesis, and wound-healing promotion attested to the comparability of the two manufacturing processes. We validated an optimized non-automated process that should allow for a GMP-compliant, more affordable, and reduced-cost strategy to exploit the potential of SVF-based regenerative therapies.

Competing Interests: Competing interests: None declared.

Competing Interests: Competing interests: None declared.

Importance: Patients with scarred vocal folds, whether congenitally or after phonosurgery, often exhibit dysphonia that negatively affects daily life and is difficult to treat. The autologous adipose tissue-derived stromal vascular fraction (ADSVF) is a readily accessible source of cells with angiogenic, anti-inflammatory, immunomodulatory, and regenerative properties., Objective: To evaluate the feasibility and tolerability of local injections of autologous ADSVF in patients with scarred vocal folds., Design, Setting, and Participants: CELLCORDES (Innovative Treatment for Scarred Vocal Cords by Local Injection of Autologous Stromal Vascular Fraction) is a prospective, open-label, single-arm, single-center, nonrandomized controlled trial with a 12-month follow-up and patient enrollment from April 1, 2016, to June 30, 2017. Eight patients with severe dysphonia attributable to vocal fold scarring associated with a congenital malformation or resulting from microsurgical sequelae (voice handicap index score >60 of 120) completed the study. Data analysis was performed from September 1, 2018, to January 1, 2019., Interventions: Injection of ADSVF into 1 or 2 vocal folds., Main Outcomes and Measures: The primary outcomes were feasibility and the number and severity of adverse events associated with ADSVF-based therapy. The secondary outcomes were changes in vocal assessment, videolaryngostroboscopy, self-evaluation of dysphonia, and quality of life at 1, 6, and 12 months after cell therapy., Results: Seven women and 1 man (mean [SD] age, 44.6 [10.4] years) were enrolled in this study. Adverse events associated with liposuction and ADSVF injection occurred; most of them resolved spontaneously. One patient received minor treatment to drain local bruising, and another experienced a minor contour defect at the liposuction site. At 12 months, the voice handicap index score was improved in all patients, with a mean (SD) improvement from baseline of 40.1 (21.5) points. Seven patients (88%) were considered to be responders, defined as improvement by 18 points or more in the voice handicap index score (the minimum clinically important difference)., Conclusions and Relevance: The findings suggest that autologous ADSVF injection in scarred vocal folds is feasible and tolerable. The findings require confirmation in a randomized clinical trial with a larger population., Trial Registration: ClinicalTrials.gov Identifier: NCT02622464.

Innovative therapies based on autologous adipose-derived stem/stromal cells (ASC) are currently being evaluated for treatment of systemic sclerosis (SSc). Although paracrine angiogenic and antifibrotic effects are considered the predominant mechanisms of ASC therapeutic potential, the impact of SSc on ASC paracrine functions remains controversial. In this study, phenotype, senescence, differentiation potential, and molecular profile were determined in ASC from SSc patients (SSc-ASC) ( n = 7) and healthy donors (HD-ASC) ( n = 7). ASC were co-cultured in indirect models with dermal fibroblasts (DF) from SSc patients or endothelial cells to assess their pro-angiogenic and antifibrotic paracrine effects. The angiogenic activity of endothelial cells was measured in vitro using tube formation and spheroid assays. DF collagen and alpha smooth muscle actin (αSMA) content were quantified after five days of co-culture with ASC. Differentiation capacity, senescence, and mRNA profiles did not differ significantly between SSc-ASC and HD-ASC. SSc-ASC retained the ability to stimulate angiogenesis through paracrine mechanisms; however, functional assays revealed reduced potential compared to HD-ASC. DF fibrosis markers were significantly decreased after co-culture with SSc-ASC. Together, these results indicate that SSc effects do not significantly compromise the angiogenic and the antifibrotic paracrine properties of ASC, thereby supporting further development of ASC-based autologous therapies for SSc treatment.

Background: In a small proportion of cases, hematopoietic function is insufficient after allogeneic hematopoietic stem cell transplantation, as a result of poor graft function or graft failure. These complications are common indications of re-mobilization of the initial donor, either for a second allograft or for an infusion of CD34+ Selected stem Cell Boost (SCB)., Methods and Materials: We retrospectively reviewed the results of two cycles of CD34+ cell mobilization and collection. CD34+ cells mobilized and collected at each cycle were compared. When CD34+ cell selection from the collected allogeneic mononuclear cells was indicated, it was performed with the Clinimacs Plus® medical device, and results from in-process and final quality checks were analyzed. To assess the efficacy of CD34+ SCB, transfusion needs before and after the infusion of selected CD34+ cells were calculated., Results: The median peripheral blood concentration of CD34+ cells/μL was marginally reduced during the second cycle (35.6 vs 33.8, p < 0.05); results revealed a strong correlation between paired values (r = 0.85). The cumulative number of collected CD34+ cells were similar for both cycles; the total processed blood volume was higher during the second cycle (p = 0.023). For CD34+ immune-selection procedures, CD34+ cell recovery and purity were respectively 57% and 95%, with a median T-cell depletion of 6.7 log. Recipients' needs for platelet and red blood cell transfusions were significantly reduced after CD34+ SCB., Conclusion: This study confirms the feasibility of a second cycle of mobilization in healthy related donors and the benefits of CD34+ SCB on hematopoietic reconstitution., (© 2019 AABB.)

Accurate characterization of hematopoietic stem cells (HSC) products is needed to better anticipate the hematopoietic reconstitution and the outcome in patients. Although CD34+ viable cells enumeration is a key predictor of time to correction of aplasia, it does not fully inform about functionality of cells contained in the graft. CFU assay is the gold standard in vitro potency assay to assess clonogenicity of HSC and consists on the count and identification of colonies several days after culture in a semi solid media. Manual count of colonies with optic microscope is the most commonly used method but its important variability and subjectivity hinders the universal implementation of this potency assay. The aim of this study is to validate a standardized method using the STEMvision™ system, the first semi-automated instrument for imaging and scoring hematopoietic colonies, according to French and European recommendations. Results obtained highlight better performance criteria with STEMvision™ system than the manual method. This semi-automatic device tends to reduce the coefficients of variation of repeatability, inter-operator variability and intermediate precision. This newly available platform could represent an interesting option, significantly improving performances of CFU assays used for the characterization of hematopoietic progenitors., (Copyright © 2019 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Objective: The autologous stromal vascular fraction (SVF) from adipose tissue is an alternative to cultured adipose-derived stem cells for use in regenerative medicine and represents a promising therapy for vasculopathy and hand disability in systemic sclerosis (SSc). However, the bioactivity of autologous SVF is not documented in this disease context. This study aimed to compare the molecular and functional profiles of the SVF-based medicinal product obtained from SSc and healthy subjects., Methods: Good manufacturing practice (GMP)-grade SVF from 24 patients with SSc and 12 healthy donors (HD) was analysed by flow cytometry to compare the distribution of the CD45- and CD45+ haematopoietic cell subsets. The ability of SVF to form a vascular network was assessed using Matrigel in vivo assay. The transcriptomic and secretory profiles of the SSc-SVF were assessed by RNA sequencing and multiplex analysis, respectively, and were compared with the HD-SVF., Results: The distribution of the leucocyte, endothelial, stromal, pericyte and transitional cell subsets was similar for SSc-SVF and HD-SVF. SSc-SVF retained its vasculogenic capacity, but the density of neovessels formed in SVF-loaded Matrigel implanted in nude mice was slightly decreased compared with HD-SVF. SSc-SVF displayed a differential molecular signature reflecting deregulation of angiogenesis, endothelial activation and fibrosis., Conclusions: Our study provides the first evidence that SSc does not compromise the vascular repair capacity of SVF, supporting its use as an innovative autologous biotherapy. The characterisation of the specific SSc-SVF molecular profile provides new perspectives for delineating markers of the potency of SVF and its targets for the treatment of SSc., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Background/aims: Platelet-rich plasma (PRP) injections are used in sports medicine and have been the subject of increased clinical interest. However, there have been very few reports of the composition of initial whole blood and the final PRP product. The objective of this study was to provide technical tools to perform a correct characterisation of platelets, leucocytes and red blood cells (RBCs) from whole blood and PRP., Methods: Blood and PRP were obtained from 26 healthy volunteers and prepared according to the varying parameters encountered within PRP process preparation and quantification (harvesting method, anticoagulant used, sampling method, counting method). Concentrations were measured at t=0, t=1, t=6  and t=24  hours., Results: Sampling of blood in Eppendorf tubes significantly decreased platelet concentration over time, whereas sampling in Microvette EDTA-coated tube kept platelet concentration stable until 24  hours. A non-significant difference was observed in platelet counts in PRP with impedance (median (IQR): 521.8  G/L (505.3-524.7)) and fluorescence (591.5  G/L (581.5-595.8)) methods. Other studied parameters did not influence platelet concentrations in blood or PRP samples. Leucocytes and RBC counts were similar whatever the anticoagulant, sampling, harvesting and counting methods used for both blood and PRP samples., Conclusions: Systematic sampling of blood and PRP in EDTA-coated tubes for quality control is recommended. The use of a validated counter for PRP sample should also be taken into account., Competing Interests: Competing interests: None declared.

Mesenchymal stem cell therapy is a promising treatment for perianal Crohn's fistulas refractory to conventional therapy, which are an extremely morbid complication and a true therapeutic challenge. Autologous adipose-derived stromal vascular fraction (ADSVF) is an easily accessible source of cells with angiogenic, anti-inflammatory, immunomodulatory, and regenerative properties. Here, we describe a case involving a patient with severe perianal Crohn's fistulas refractory to the best medical and surgical practices who received local treatment with ADSVF and microfat. This patient was first examined under anesthesia with drainage via seton placement; 1 week later, on a single day, he underwent adipose tissue extraction, ADSVF and microfat preparation, and the local injection of 14 ml of microfat and approximately 20 million viable ADSVF cells into the soft tissue around the fistulas. No serious adverse events were observed. At the first endpoint at 12 weeks, the fistula had clinically healed with complete re-epithelialization of all external openings; no fistula tract was detected on magnetic resonance imaging, confirming this finding. This good clinical outcome was sustained at 48 weeks and was associated with a reduction in the severity of perianal disease and an improvement in quality of life. The current case highlights the therapeutic potential of a new cellular treatment for Crohn's patients with refractory perianal fistulas based on the innovative hypothesis that the combined action of ADSVF in association with the trophic characteristics of a microfat graft could be beneficial for this condition., Trial Registration: EudraCT number 201325, NCT02520843 . Registered on 5 August 2015.