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.

Angiotensin II (AngII), the main effector peptide of the renin-angiotensin system (RAS), participates in multiple biological processes, including cell growth, apoptosis, and tissue remodeling. Since AngII activates, in different cell types, signal transducing pathways that are critical for mammary gland postlactational regression, we investigated the role of the RAS during this process. We found that exogenous administration of AngII in mammary glands of lactating Balb/c mice induced epithelium apoptosis [2.9±0.5% (control) vs. 9.6±1.1% (AngII); P < 0.001] and activation of the proapoptotic factor STAT3, an effect inhibited by irbesartan, an AT(1) receptor blocker. Subsequently, we studied the expression kinetics of RAS components during involution. We found that angiotensin-converting enzyme (ACE) mRNA expression peaked 6 h after weaning (5.7-fold; P<0.01), while induction of angiotensinogen and AT(1) and AT(2) receptors expression was detected 96 h after weaning (6.2-, 10-, and 6.2-fold increase, respectively; P<0.01). To assess the role of endogenously generated AngII, mice were treated with losartan, an AT(1) receptor blocker, during mammary involution. Mammary glands from losartan-treated mice showed activation of the survival factors AKT and BCL-(XL), significantly lower LIF and TNF-α mRNA expression (P<0.05), reduced apoptosis [12.1±2.1% (control) vs. 4.8±0.7% (losartan); P<0.001] and shedding of epithelial cells, inhibition of MMP-9 activity in a dose-dependent manner (80%; P<0.05; with losartan IC(50) value of 6.9 mg/kg/d] and lower collagen deposition and adipocyte invasion causing a delayed involution compared to vehicle-treated mice. Furthermore, mammary glands of forced weaned AT(1A)- and/or AT(1B)-deficient mice exhibited retarded apoptosis of epithelial cells [6.3±0.95% (WT) vs. 3.3±0.56% (AT(1A)/AT(1B) DKO); P<0.05] with remarkable delayed postlactational regression compared to wild-type animals. Taken together, these results strongly suggest that AngII, via the AT(1) receptor, plays a major role in mouse mammary gland involution identifying a novel role for the RAS. angiotensin system.