The glycoprotein gO (UL74) of human cytomegalovirus (HCMV) forms a complex with gH/gL. Virus mutants with a deletion of gO show a defect in secondary envelopment with the consequence that virus spread is restricted to a cell-associated pathway. Here we report that the positional homolog of HCMV gO, m74 of mouse CMV (MCMV), codes for a glycosylated protein which also forms a complex with gH (M75). m74 knockout mutants of MCMV show the same spread phenotype as gO knockout mutants of HCMV, namely, a shift from supernatant-driven to cell-associated spread. We could show that this phenotype is due to a reduction of infectious virus particles in cell culture supernatants. m74 knockout mutants enter fibroblasts via an energydependent and pH-sensitive pathway, whereas in the presence of an intact m74 gene product, entry is neither energy dependent nor pH sensitive. This entry phenotype is shared by HCMV expressing or lacking gO. Our data indicate that the m74 and UL74 gene products both codetermine CMV spread and CMV entry into cells. We postulate that MCMV, like HCMV, expresses alternative gH/gL complexes which govern cell-to-cell spread of the virus.

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, 32À100%) 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.

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.

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.

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