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Corneal transparency and avascularity are essential for vision. The avascular cornea transitions into the vascularized conjunctiva at the limbus. Here, we explore a limbal stromal cell sub-population that expresses ABCB5 and has mesenchymal stem cell characteristics. Human primary corneal stromal cells were enriched for ABCB5 by using FACS sorting. ABCB5+ cells expressed the MSC markers CD90, CD73, and CD105. ABCB5+ but not ABCB5- cells from the same donor displayed evidence of pluripotency with a significantly higher colony-forming efficiency and the ability of trilineage differentiation (osteogenic, adipogenic, and chondrogenic). The ABCB5+ cell secretome demonstrated lower levels of the pro-inflammatory protein MIF (macrophage migration inhibitory factor) as well as of the pro-(lymph)angiogenic growth factors VEGFA and VEGFC, which correlated with reduced proliferation of Jurkat cells co-cultured with ABCB5+ cells and decreased proliferation of blood and lymphatic endothelial cells cultured in ABCB5+ cell-conditioned media. These data support the hypothesis that ABCB5+ limbal stromal cells are a putative MSC population with potential anti-inflammatory and anti-(lymph)angiogenic effects. The therapeutic modulation of ABCB5+ limbal stromal cells may prevent cornea neovascularization and inflammation and, if transplanted to other sites in the body, provide similar protective properties to other tissues.

Background: Given the high level of product complexity and limited regulatory guidance, designing and implementing appropriate potency assays is often the most challenging part of establishing a quality control testing matrix for a cell-based medicinal product. Among the most elusive tasks are the selection of suitable read-out parameters, the development of assay designs that most closely model the pathophysiological conditions, and the validation of the methods. Here we describe these challenges and how they were addressed in developing an assay that measures the anti-inflammatory potency of mesenchymal stromal cells (MSCs) in an M1 macrophage-dominated inflammatory environment., Methods: An in vitro inflammation model was established by coculturing skin-derived ABCB5 + MSCs with THP-1 monocyte-derived M1-polarized macrophages. Readout was the amount of interleukin 1 receptor antagonist (IL-1RA) secreted by the MSCs in the coculture, measured by an enzyme-linked immunosorbent assay., Results: IL-1RA was quantified with guideline-concordant selectivity, accuracy and precision over a relevant concentration range. Consistent induction of the macrophage markers CD36 and CD80 indicated successful macrophage differentiation and M1 polarization of THP-1 cells, which was functionally confirmed by release of proinflammatory tumor necrosis factor α. Testing a wide range of MSC/macrophage ratios revealed the optimal ratio for near-maximal stimulation of MSCs to secrete IL-1RA, providing absolute maximum levels per individual MSC that can be used for future comparison with clinical efficacy. Batch release testing of 71 consecutively manufactured MSC batches showed a low overall failure rate and a high comparability between donors., Conclusions: We describe the systematic development and validation of a therapeutically relevant, straightforward, robust and reproducible potency assay to measure the immunomodulatory capacity of MSCs in M1 macrophage-driven inflammation. The insights into the challenges and how they were addressed may also be helpful to developers of potency assays related to other cellular functions and clinical indications., Competing Interests: Declaration of Competing of interest MHF is inventor or coinventor of U.S. and international patents assigned to Brigham and Women's Hospital and/or Boston Children's Hospital, licensed to RHEACELL GmbH & Co. KG. MHF and KS-K serve as scientific advisors to RHEACELL. MHF holds stock in RHEACELL. SS, LN, EN-R, AN, SH, LV and JE are employees of RHEACELL. CG is CEO, and MAK is COO of RHEACELL., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Acute kidney injury (AKI) is characterized by a rapid reduction in renal function and glomerular filtration rate (GFR). The broadly used anti-cancer chemotherapeutic agent cisplatin often induces AKI as an adverse drug side effect. Therapies targeted at the reversal of AKI and its potential progression to chronic kidney disease or end-stage renal disease are currently insufficiently effective. Mesenchymal stromal cells (MSCs) possess diverse immunomodulatory properties that confer upon them significant therapeutic potential for the treatment of diverse inflammatory disorders. Human dermal MSCs expressing ATP-Binding Cassette member B5 (ABCB5) have shown therapeutic efficacy in clinical trials in chronic skin wounds or recessive dystrophic epidermolysis bullosa. In preclinical studies, ABCB5+ MSCs have also shown to reverse metabolic reprogramming in polycystic kidney cells, suggesting a capacity for this cell subset to improve also organ function in kidney diseases. Here, we aimed to explore the therapeutic capacity of ABCB5+ MSCs to improve renal function in a preclinical rat model of cisplatin-induced AKI. First, the anti-apoptotic and immunomodulatory capacity was compared against research-grade adipose stromal cells (ASCs). Then, cross-species immunomodulatory capacity was checked, testing first inhibition of mitogen-driven peripheral blood mononuclear cells and then modulation of macrophage function. Finally, therapeutic efficacy was evaluated in a cisplatin AKI model. First, ABCB5+ MSCs suppressed cisplatin-induced apoptosis of human conditionally-immortalized proximal tubular epithelial cells in vitro , most likely by reducing oxidative stress. Second, ABCB5+ MSCs inhibited the proliferation of either human or rat peripheral blood mononuclear cells, in the human system via the Indoleamine/kynurenine axis and in the murine context via nitric oxide/nitrite. Third, ABCB5+ MSCs decreased TNF-α secretion after lipopolysaccharide stimulation and modulated phagocytosis and in both human and rat macrophages, involving prostaglandin E2 and TGF-β1, respectively. Fourth, clinical-grade ABCB5+ MSCs grafted intravenously and intraperitoneally to a cisplatin-induced AKI murine model exerted modulatory effects on mRNA expression patterns toward an anti-inflammatory and pro-regenerative state despite an apparent lack of amelioration of renal damage at physiologic, metabolic, and histologic levels. Our results demonstrate anti-inflammatory and pro-regenerative effects of clinical grade ABCB5+ MSCs in vitro and in vivo and suggest potential therapeutic utility of this cell population for treatment or prevention of cisplatin chemotherapy-induced tissue toxicity., Competing Interests: MK is CSO of RHEACELL. CD is project manager of RHEACELL GmbH & Co. KG, Heidelberg, Germany. CG is Managing Partner and holds equity in RHEACELL GmbH & Co. KG. MF is inventor or co-inventor of US and international patents assigned to Brigham and Women’s Hospital and/or Boston Children’s Hospital and licensed to RHEACELL GmbH & Co. KG Heidelberg, Germany. He also serves as a scientific advisor to and holds equity in RHEACELL GmbH & Co. KG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Rendra, Crigna, Daniele, Sticht, Cueppers, Kluth, Ganss, Frank, Gretz and Bieback.)

Adipose-derived stem cells (ASCs) have been used as a therapeutic intervention for peripheral artery disease (PAD) in clinical trials. To further explore the therapeutic mechanism of these mesenchymal multipotent stromal/stem cells in PAD, this study was designed to test the effect of xenogeneic ASCs extracted from human adipose tissue on hypoxic endothelial cells (ECs) and terminal unfolded protein response (UPR) in vitro and in an atherosclerosis-prone apolipoprotein E-deficient mice (ApoE -/- mice) hindlimb ischemia model in vivo. ASCs were added to Cobalt (II) chloride-treated ECs; then, metabolic activity, cell migration, and tube formation were evaluated. Fluorescence-based sensors were used to assess dynamic changes in Ca 2+ levels in the cytosolic- and endoplasmic reticulum (ER) as well as changes in reactive oxygen species. Western blotting was used to observe the UPR pathway. To simulate an acute-on-chronic model of PAD, ApoE -/- mice were subjected to a double ligation of the femoral artery (DLFA). An assessment of functional recovery after DFLA was conducted, as well as histology of gastrocnemius. Hypoxia caused ER stress in ECs, but ASCs reduced it, thereby promoting cell survival. Treatment with ASCs ameliorated the effects of ischemia on muscle tissue in the ApoE -/- mice hindlimb ischemia model. Animals showed less muscle necrosis, less inflammation, and lower levels of muscle enzymes after ASC injection. In vitro and in vivo results revealed that all ER stress sensors (BIP, ATF6, CHOP, and XBP1) were activated. We also observed that the expression of these proteins was reduced in the ASCs treatment group. ASCs effectively alleviated endothelial dysfunction under hypoxic conditions by strengthening ATF6 and initiating a transcriptional program to restore ER homeostasis. In general, our data suggest that ASCs may be a meaningful treatment option for patients with PAD who do not have traditional revascularization options.

Quantitative polymerase chain reaction (qPCR) has emerged as an important bioanalytical method for assessing the pharmacokinetics of human-cell-based medicinal products after xenotransplantation into immunodeficient mice. A particular challenge in bioanalytical qPCR studies is that the different tissues of the host organism can affect amplification efficiency and amplicon detection to varying degrees, and ignoring these matrix effects can easily cause a significant underestimation of the true number of target cells in a sample. Here, we describe the development and drug regulatory-compliant validation of a TaqMan ® qPCR assay for the quantification of mesenchymal stromal cells in the range of 125 to 20,000 cells/200 µL lysate via the amplification of a human-specific, highly repetitive α-satellite DNA sequence of the chromosome 17 centromere region HSSATA17. An assessment of matrix effects in 14 different mouse tissues and blood revealed a wide range of spike recovery rates across the different tissue types, from 11 to 174%. Based on these observations, we propose performing systematic spike-and-recovery experiments during assay validation and correcting for the effects of the different tissue matrices on cell quantification in subsequent bioanalytical studies by multiplying the back-calculated cell number by tissue-specific factors derived from the inverse of the validated percent recovery rate.

Background and Aims: Recessive dystrophic epidermolysis bullosa (RDEB) is a hereditary, rare, devastating and life-threatening skin fragility disorder with a high unmet medical need. In a recent international, single-arm clinical trial, treatment of 16 patients (aged 6-36 years) with three intravenous infusions of 2 × 10 6  immunomodulatory ABCB5 + dermal mesenchymal stromal cells (MSCs)/kg on days 0, 17 and 35 reduced disease activity, itch and pain. A post-hoc analysis was undertaken to assess the potential effects of treatment with ABCB5 + MSCs on the overall skin wound healing in patients suffering from RDEB., Methods: Documentary photographs of the affected body regions taken on days 0, 17, 35 and at 12 weeks were evaluated regarding proportion, temporal course and durability of wound closure as well as development of new wounds., Results: Of 168 baseline wounds in 14 patients, 109 (64.9%) wounds had closed at week 12, of which 63.3% (69 wounds) had closed already by day 35 or day 17. Conversely, 74.2% of the baseline wounds that had closed by day 17 or day 35 remained closed until week 12. First-closure ratio within 12 weeks was 75.6%. The median rate of newly developing wounds decreased significantly (P = 0.001) by 79.3%., Conclusions: Comparison of the findings with published data from placebo arms and vehicle-treated wounds in controlled clinical trials suggests potential capability of ABCB5 + MSCs to facilitate wound closure, prolongate wound recurrence and decelerate formation of new wounds in RDEB. Beyond suggesting therapeutic efficacy for ABCB5 + MSCs, the analysis might stimulate researchers who develop therapies for RDEB and other skin fragility disorders to not only assess closure of preselected target wounds but pay attention to the patients' dynamic and diverse overall wound presentation as well as to the durability of achieved wound closure and the development of new wounds., Trial Registration: Clinicaltrials.gov NCT03529877; EudraCT 2018-001009-98., Competing Interests: Declaration of Competing Interest NYF and MHF are inventors or co-inventors of U.S. and international patents assigned to Brigham and Women's Hospital, Boston Children's Hospital, and/or the VA Boston Healthcare System, Boston, Massachusetts, USA, licensed to TICEBA GmbH, Heidelberg, Germany, and RHEACELL GmbH & Co. KG, Heidelberg, Germany. MHF serves as scientific advisor and holds stock in TICEBA GmbH and RHEACELL GmbH & Co. KG. DK and JT serve as scientific advisors to RHEACELL. KD, CD and SF are employees of RHEACELL. ENR is employee of TICEBA. CG is CEO, and MAK is CSO of TICEBA and RHEACELL. JAM declares that he has no conflicts of interest., (Copyright © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Recessive dystrophic epidermolysis (RDEB) is a rare, inherited, and currently incurable skin blistering disorder characterized by cyclically recurring wounds coexisting with chronic non-healing wounds. In a recent clinical trial, three intravenous infusions of skin-derived ABCB5 + mesenchymal stromal cells (MSCs) to 14 patients with RDEB improved the healing of wounds that were present at baseline. Since in RDEB even minor mechanical forces perpetually provoke the development of new or recurrent wounds, a post-hoc analysis of patient photographs was performed to specifically assess the effects of ABCB5 + MSCs on new or recurrent wounds by evaluating 174 wounds that occurred after baseline. During 12 weeks of systemic treatment with ABCB5 + MSCs, the number of newly occurring wounds declined. When compared to the previously reported healing responses of the wounds present at baseline, the newly occurring wounds healed faster, and a greater portion of healed wounds remained stably closed. These data suggest a previously undescribed skin-stabilizing effect of treatment with ABCB5 + MSCs and support repeated dosing of ABCB5 + MSCs in RDEB to continuously slow the wound development and accelerate the healing of new or recurrent wounds before they become infected or progress to a chronic, difficult-to-heal stage.

Background: Hypoxia in ischemic disease impairs Ca 2+ homeostasis and may promote angiogenesis. The therapeutic efficacy of mesenchymal stromal cells (MSCs) in peripheral arterial occlusive disease is well established, yet its influence on cellular Ca 2+ homeostasis remains to be elucidated. We addressed the influence of ATP-binding cassette subfamily B member 5 positive mesenchymal stromal cells (ABCB5 + MSCs) on Ca 2+ homeostasis in hypoxic human umbilical vein endothelial cells (HUVECs) in vitro and in vivo., Methods: Hypoxia was induced in HUVECs by Cobalt (II) chloride (CoCl 2 ) or Deferoxamine (DFO). Dynamic changes in the cytosolic- and endoplasmic reticulum (ER) Ca 2+ and changes in reactive oxygen species were assessed by appropriate fluorescence-based sensors. Metabolic activity, cell migration, and tube formation were assessed by standard assays. Acute-on-chronic ischemia in Apolipoprotein E knock-out (ApoE -/- ) mice was performed by double ligation of the right femoral artery (DFLA). ABCB5 + MSC cells were injected into the ischemic limb. Functional recovery after DFLA and histology of gastrocnemius and aorta were assessed., Results: Hypoxia-induced impairment of cytosolic and ER Ca 2+ were restored by ABCB5 + MSCs or their conditioned medium. Similar was found for changes in intracellular ROS production, metabolic activity, migratory ability and tube formation. The restoration was paralleled by an increased expression of the Ca 2+ transporter Sarco-/endoplasmic reticulum ATPase 2a (SERCA2a) and the phosphorylation of Phospholamban (PLN). In acute-on-chronic ischemia, ABCB5 + MSCs treated mice showed a higher microvascular density, increased SERCA2a expression and PLN phosphorylation relative to untreated controls., Conclusions: ABCB5 + MSCs therapy can restore cellular Ca 2+ homeostasis, which may beneficially affect the angiogenic function of endothelial cells under hypoxia in vitro and in vivo., (© 2023. The Author(s).)

The ATP-binding cassette superfamily member ABCB5 identifies a subset of skin-resident mesenchymal stem cells (MSCs) that exhibit potent immunomodulatory and wound healing-promoting capacities along with superior homing ability. The ABCB5 + MSCs can be easily accessed from discarded skin samples, expanded, and delivered as a highly homogenous medicinal product with standardized potency. A range of preclinical studies has suggested therapeutic efficacy of ABCB5 + MSCs in a variety of currently uncurable skin and non-skin inflammatory diseases, which has been substantiated thus far by distinct clinical trials in chronic skin wounds or recessive dystrophic epidermolysis bullosa. Therefore, skin-derived ABCB5 + MSCs have the potential to provide a breakthrough at the forefront of MSC-based therapies striving to fulfill current unmet medical needs. The most recent milestones in this regard are the approval of a phase III pivotal trial of ABCB5 + MSCs for treatment of recessive dystrophic and junctional epidermolysis bullosa by the US Food and Drug Administration, and national market access of ABCB5 + MSCs (AMESANAR ® ) for therapy-refractory chronic venous ulcers under the national hospital exemption pathway in Germany.

Background: While rapid healing of diabetic foot ulcers (DFUs) is highly desirable to avoid infections, amputations and life-threatening complications, DFUs often respond poorly to standard treatment. GMP-manufactured skin-derived ABCB5 + mesenchymal stem cells (MSCs) might provide a new adjunctive DFU treatment, based on their remarkable skin wound homing and engraftment potential, their ability to adaptively respond to inflammatory signals, and their wound healing-promoting efficacy in mouse wound models and human chronic venous ulcers., Methods: The angiogenic potential of ABCB5 + MSCs was characterized with respect to angiogenic factor expression at the mRNA and protein level, in vitro endothelial trans-differentiation and tube formation potential, and perfusion-restoring capacity in a mouse hindlimb ischemia model. Finally, the efficacy and safety of ABCB5 + MSCs for topical adjunctive treatment of chronic, standard therapy-refractory, neuropathic plantar DFUs were assessed in an open-label single-arm clinical trial., Results: Hypoxic incubation of ABCB5 + MSCs led to posttranslational stabilization of the hypoxia-inducible transcription factor 1α (HIF-1α) and upregulation of HIF-1α mRNA levels. HIF-1α pathway activation was accompanied by upregulation of vascular endothelial growth factor (VEGF) transcription and increase in VEGF protein secretion. Upon culture in growth factor-supplemented medium, ABCB5 + MSCs expressed the endothelial-lineage marker CD31, and after seeding on gel matrix, ABCB5 + MSCs demonstrated formation of capillary-like structures comparable with human umbilical vein endothelial cells. Intramuscularly injected ABCB5 + MSCs to mice with surgically induced hindlimb ischemia accelerated perfusion recovery as measured by laser Doppler blood perfusion imaging and enhanced capillary proliferation and vascularization in the ischemic muscles. Adjunctive topical application of ABCB5 + MSCs onto therapy-refractory DFUs elicited median wound surface area reductions from baseline of 59% (full analysis set, n = 23), 64% (per-protocol set, n = 20) and 67% (subgroup of responders, n = 17) at week 12, while no treatment-related adverse events were observed., Conclusions: The present observations identify GMP-manufactured ABCB5 + dermal MSCs as a potential, safe candidate for adjunctive therapy of otherwise incurable DFUs and justify the conduct of a larger, randomized controlled trial to validate the clinical efficacy., Trial Registration: ClinicalTrials.gov, NCT03267784, Registered 30 August 2017, https://clinicaltrials.gov/ct2/show/NCT03267784., (© 2022. The Author(s).)

BACKGROUNDRecessive dystrophic epidermolysis bullosa (RDEB) is a rare, devastating, and life-threatening inherited skin fragility disorder that comes about due to a lack of functional type VII collagen, for which no effective therapy exists. ABCB5+ dermal mesenchymal stem cells (ABCB5+ MSCs) possess immunomodulatory, inflammation-dampening, and tissue-healing capacities. In a Col7a1-/- mouse model of RDEB, treatment with ABCB5+ MSCs markedly extended the animals' lifespans.METHODSIn this international, multicentric, single-arm, phase I/IIa clinical trial, 16 patients (aged 4-36 years) enrolled into 4 age cohorts received 3 i.v. infusions of 2 × 106 ABCB5+ MSCs/kg on days 0, 17, and 35. Patients were followed up for 12 weeks regarding efficacy and 12 months regarding safety.RESULTSAt 12 weeks, statistically significant median (IQR) reductions in the Epidermolysis Bullosa Disease Activity and Scarring Index activity (EBDASI activity) score of 13.0% (2.9%-30%; P = 0.049) and the Instrument for Scoring Clinical Outcome of Research for Epidermolysis Bullosa clinician (iscorEB‑c) score of 18.2% (1.9%-39.8%; P = 0.037) were observed. Reductions in itch and pain numerical rating scale scores were greatest on day 35, amounting to 37.5% (0.0%-42.9%; P = 0.033) and 25.0% (-8.4% to 46.4%; P = 0.168), respectively. Three adverse events were considered related to the cell product: 1 mild lymphadenopathy and 2 hypersensitivity reactions. The latter 2 were serious but resolved without sequelae shortly after withdrawal of treatment.CONCLUSIONThis trial demonstrates good tolerability, manageable safety, and potential efficacy of i.v. ABCB5+ MSCs as a readily available disease-modifying therapy for RDEB and provides a rationale for further clinical evaluation.TRIAL REGISTRATIONClinicaltrials.gov NCT03529877; EudraCT 2018-001009-98.FUNDINGThe trial was sponsored by RHEACELL GmbH & Co. KG. Contributions by NYF and MHF to this work were supported by the NIH/National Eye Institute (NEI) grants RO1EY025794 and R24EY028767.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare, incurable blistering skin disease caused by biallelic mutations in type VII collagen (C7). Advancements in treatment of RDEB have come from harnessing the immunomodulatory potential of mesenchymal stem cells (MSCs). Although human bone marrow-derived MSC (BM-MSC) trials in RDEB demonstrate improvement in clinical severity, the mechanisms of MSC migration to and persistence in injured skin and their contributions to wound healing are not completely understood. A unique subset of MSCs expressing ATP-binding cassette subfamily member 5 (ABCB5) resides in the reticular dermis and exhibits similar immunomodulatory characteristics to BM-MSCs. Our work aimed to test the hypothesis that skin-derived ABCB5+ dermal MSCs (DSCs) possess superior skin homing ability compared to BM-MSCs in immunodeficient NOD-scid IL2rgamma null (NSG) mice. Compared to BM-MSCs, peripherally injected ABCB5+ DSCs demonstrated superior homing and engraftment of wounds. Furthermore, ABCB5+ DSCs vs BM-MSCs cocultured with macrophages induced less anti-inflammatory interleukin-1 receptor antagonist (IL-1RA) production. RNA sequencing of ABCB5+ DSCs compared to BM-MSCs showed unique expression of major histocompatibility complex class II and Homeobox (Hox) genes, specifically HOXA3. Critical to inducing migration of endothelial and epithelial cells for wound repair, increased expression of HOXA3 may explain superior skin homing properties of ABCB5+ DSCs. Further discernment of the immunomodulatory mechanisms among MSC populations could have broader regenerative medicine implications beyond RDEB treatment., (© 2021 AlphaMed Press.)

Background Aim: Mesenchymal stromal cells (MSCs) hold promise for the treatment of tissue damage and injury. However, MSCs comprise multiple subpopulations with diverse properties, which could explain inconsistent therapeutic outcomes seen among therapeutic attempts. Recently, the adenosine triphosphate-binding cassette transporter ABCB5 has been shown to identify a novel dermal immunomodulatory MSC subpopulation., Methods: The authors have established a validated Good Manufacturing Practice (GMP)-compliant expansion and manufacturing process by which ABCB5 + MSCs can be isolated from skin tissue and processed to generate a highly functional homogeneous cell population manufactured as an advanced therapy medicinal product (ATMP). This product has been approved by the German competent regulatory authority to be tested in a clinical trial to treat therapy-resistant chronic venous ulcers., Results: As of now, 12 wounds in nine patients have been treated with 5 × 10 5  autologous ABCB5 + MSCs per cm 2 wound area, eliciting a median wound size reduction of 63% (range, 32-100%) at 12 weeks and early relief of pain., Conclusions: The authors describe here their GMP- and European Pharmacopoeia-compliant production and quality control process, report on a pre-clinical dose selection study and present the first in-human results. Together, these data substantiate the idea that ABCB5 + MSCs manufactured as ATMPs could deliver a clinically relevant wound closure strategy for patients with chronic therapy-resistant wounds., (Copyright © 2020 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Human dermal mesenchymal stromal cells (MSCs) expressing the ATP-binding cassette (ABC) efflux transporter ABCB5 represent an easily accessible MSC population that, based on preclinical and first-in-human data, holds significant promise to treat a broad spectrum of conditions associated not only with skin-related but also systemic inflammatory and/or degenerative processes., Methods: We have developed a validated Good Manufacturing Practice-compliant expansion and manufacturing process by which ABCB5 + MSCs derived from surgical discard skin tissues are processed to an advanced-therapy medicinal product (ATMP) for clinical use. Enrichment for ABCB5 + MSCs is achieved in a three-step process involving plastic adherence selection, expansion in a highly efficient MSC-selecting medium, and immunomagnetic isolation of the ABCB5 + cells from the mixed culture., Results: Product Quality Review data covering 324 cell expansions, 728 ABCB5 + MSC isolations, 66 ABCB5 + MSC batches, and 85 final drug products reveal high process robustness and reproducible, reliable quality of the manufactured cell therapy product., Conclusion: We have successfully established an expansion and manufacturing process that enables the generation of homogenous ABCB5 + MSC populations of proven biological activity manufactured as a standardized, donor-independent, highly pure, and highly functional off-the-shelf available ATMP, which is currently tested in multiple clinical trials.

Although liver transplantation is a potential effective cure for patients with end-stage liver diseases, this strategy has several drawbacks including high cost, long waiting list, and limited availability of liver organs. Therefore, stem cell-based therapy is presented as an alternative option, which showed promising results in animal models of acute and chronic liver injuries. ABCB5 + cells isolated from skin dermis represent an easy accessible and expandable source of homogenous stem cell populations. In addition, ABCB5 + cells showed already promising results in the treatment of corneal and skin injury. To date, the effect of these cells on liver injury is still unknown. In the current study, sixteen weeks old Mdr2KO mice were i.v. injected with 500,000 ABCB5 + cells using different experimental setups. The effects of cellular therapy on inflammation, fibrosis, apoptosis, and proliferation were analyzed in the collected liver tissues. Toxicity of ABCB5 + cells was additionally investigated in mice with partial liver resection. In vitro, the fibrosis- and inflammatory-modulating effects of supernatant from ABCB5 + cells were examined in the human hepatic stellate cell line (LX-2). Cell injections into fibrotic Mdr2KO mice as well as into mice upon partial liver resection have no signs of toxicity with regard to cell transformation, cellular damage, fibrosis or inflammation as compared to controls. We next investigated the effects of ABCB5 + cells on established biliary liver fibrosis in the Mdr2KO mice. ABCB5 + cells to some extent influenced the shape of the liver inflammatory response and significantly reduced the amount of collagen deposition, as estimated from quantification of sirius red staining. Furthermore, reduced apoptosis and enhanced death compensatory proliferation resulted from ABCB5 + cell transformation. The stem cells secreted several trophic factors that activated TGF-β family signaling in cultured LX-2 hepatic stellate cells (HSCs), therewith shaping cell fate to an αSMA high , Vimentin low phenotype. Taken together, ABCB5 + cells can represent a safe and feasible strategy to support liver regeneration and to reduce liver fibrosis in chronic liver diseases.

In this study, we report the beneficial effects of a newly identified dermal cell subpopulation expressing the ATP-binding cassette subfamily B member 5 (ABCB5) for the therapy of nonhealing wounds. Local administration of dermal ABCB5 + -derived mesenchymal stem cells (MSCs) attenuated macrophage-dominated inflammation and thereby accelerated healing of full-thickness excisional wounds in the iron-overload mouse model mimicking the nonhealing state of human venous leg ulcers. The observed beneficial effects were due to interleukin-1 receptor antagonist (IL-1RA) secreted by ABCB5 + -derived MSCs, which dampened inflammation and shifted the prevalence of unrestrained proinflammatory M1 macrophages toward repair promoting anti-inflammatory M2 macrophages at the wound site. The beneficial anti-inflammatory effect of IL-1RA released from ABCB5 + -derived MSCs on human wound macrophages was conserved in humanized NOD-scid IL2rγ null mice. In conclusion, human dermal ABCB5 + cells represent a novel, easily accessible, and marker-enriched source of MSCs, which holds substantial promise to successfully treat chronic nonhealing wounds in humans. Stem Cells 2019;37:1057-1074., (© 2019 The Authors. Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press 2019.)

Background Aims: Human dermal ABCB5-expressing mesenchymal stromal cells (ABCB5 + MSCs) represent a promising candidate for stem cell-based therapy of various currently uncurable diseases in several fields of regenerative medicine. We have developed and validated a method to isolate, from human skin samples, and expand ABCB5 + MSCs that meet the guideline criteria of the International Society for Cellular Therapy. We are able to process these cells into a Good Manufacturing Practice-conforming, MSC-based advanced-therapy medicinal product., Methods: To support the development of ABCB5 + MSCs for potential therapeutic topical, intramuscular and intravenous administration, we have tested our product in a series of Good Laboratory Practice-compliant nonclinical in-vivo studies addressing all relevant aspects of biosafety, including potential long-term persistence and proliferation, distribution to nontarget tissues, differentiation into undesired cell types, ectopic tissue formation, tumor formation and local tissue reaction., Results: (i) Subcutaneous application of 1 × 10 7 ABCB5 + MSCs/animal and intravenous application of 2 × 10 6 ABCB5 + MSCs/animal, respectively, to immunocompromised mice did not result in safety-relevant biodistribution, persistence or proliferation of the cells; (ii) three monthly subcutaneous injections of ABCB5 + MSCs at doses ranging from 1 × 10 5 to 1 × 10 7 cells/animal and three biweekly intravenous injections of 2 × 10 6 ABCB5 + MSCs/animal, respectively, to immunocompromised mice were nontoxic and revealed no tumorigenic potential; and (iii) intramuscular injection of 5 × 10 6 ABCB5 + MSCs/animal to immunocompromised mice was locally well tolerated., Discussion: The present preclinical in vivo data demonstrate the local and systemic safety and tolerability of a novel advanced-therapy medicinal product based on human skin-derived ABCB5 + MSCs., (Copyright © 2019 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)

The continuously decreasing willingness for liver donation aggravates treatment of end-stage liver diseases requiring organ transplantation as the only curative strategy. Cell therapy approaches using human hepatocytes or stem cell-derived hepatocyte-like cells may be a therapeutic option out of this dilemma. ABCB5-positive mesenchymal stromal cells from human skin featured promising potential to treat immune-mediated diseases. Since most of chronic liver diseases involve exaggerating immune mechanisms, it was the aim to demonstrate in this study, whether ABCB5 + stem cells may serve as a resource to generate hepatocytic cells for application in liver cell transplantation. Using an established single-step protocol, which had been successfully applied to differentiate mesenchymal stromal cells into the hepatocytic lineage, ABCB5 + skin-derived stem cells did not gain significant characteristics of hepatocytes. Yet, upon culture in hepatocytic differentiation medium, ABCB5 + stem cells secreted immunomodulatory and anti-fibrotic factors as well as proteins, which may prompt hepatic morphogenesis besides others. Hepatic transplantation of ABCB5 + stem cells, which had been prior cultured in hepatocytic differentiation medium, did not cause any obvious deterioration of liver architecture suggesting their safe application. Thus, human ABCB5 + skin-derived stem cells secreted putative hepatotropic factors after culture in hepatocytic differentiation medium., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mesenchymal stem cells (MSCs) are crucial for tissue homeostasis and regeneration. Though of prime interest, their potentially protective role on neutrophil-induced tissue damage, associated with high morbidity and mortality, has not been explored in sufficient detail. Here we report the therapeutic skill of MSCs to suppress unrestrained neutrophil activation and to attenuate severe tissue damage in a murine immune-complex mediated vasculitis model of unbalanced neutrophil activation. MSC-mediated neutrophil suppression was due to intercellular adhesion molecule 1-dependent engulfment of neutrophils by MSCs, decreasing overall neutrophil numbers. Similar to MSCs in their endogenous niche of murine and human vasculitis, therapeutically injected MSCs via upregulation of the extracellular superoxide dismutase (SOD3), reduced superoxide anion concentrations and consequently prevented neutrophil death, neutrophil extracellular trap formation and spillage of matrix degrading neutrophil elastase, gelatinase and myeloperoxidase. SOD3-silenced MSCs did not exert tissue protective effects. Thus, MSCs hold substantial therapeutic promise to counteract tissue damage in conditions with unrestrained neutrophil activation. Stem Cells 2016;34:2393-2406., (© 2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)