Lanza, F. | Search bioRxiv

bioRxiv2022

Efficacy and safety of a patch containing adipose-derived stem cells for skin wound healing. Results form a comprehensive pre-clinical evaluation program

Mesenchymal stem cell-based therapies are emerging as innovative approaches to treat chronic wounds. A common administration route used in clinical trials consists of local injections leading to uncontrolled/sub-optimal delivery. This study reports a comprehensive pre-clinical evaluation program on the mechanism of action, efficacy and safety of an easy-to-use patch that concentrates Adipose-derived Stem Cells (ASCs) in a clinical-grade sponge of porcine crosslinked-gelatin. ASCs were prepared from the fat of ischemic patients. Transcriptome and proteome of ASC-patches and ASC monolayers were assessed by microarrays, bio-arrays and mass spectrometry. Tumorigenesis was investigated in immunosuppressed mice according to the European Pharmacopeia. Angiogenesis was assessed in vivo in the chick chorioallantoic membrane model. Efficacy of the ASC-patch was tested in a rat model of ischemic full-thickness skin defect. Cell stability was assessed by luminescence using ASC-patches generated from ASCs stably transduced with firefly luciferase. ASCs from ischemic patients upregulated the transcription of multiple genes involved in skin wound healing when cultured within the ASC-patch formulation. The patch was not only a concentrator, but also a reservoir of both ASC-derived regenerative factors and sponge-derived soluble fragments having healing capacity. The secretome of the ASC-patch promoted dermal fibroblast survival and epidermal epithelialization. No tumor formation was observed in immunodeficient Nude mice subcutaneously transplanted with the ASC-patch. Transplanted patches were early invaded by new vessels in vivo, and a marked angiogenesis was confirmed in two independent animal models. Finally, ASC-patches prepared from syngeneic rats promoted faster healing and re-vascularization of full-thickness skin defects in a rat animal model. Of note, ASCs were viable and locally stable for at least two weeks in vivo. We provide here compelling pre-clinical evidence that patches concentrating ASCs within crosslinked gelatin may represent a convenient and effective tool for the management of chronic wounds.

bioengineering↗

bioRxiv2021

Transcriptome analysis of the effect of AHR on productive and unproductive pathways of in vitro megakaryocytopoiesis

Human CD34+ progenitors can be differentiated in vitro into proplatelet-producing megakaryocytes (MKs) within 17 days. During this time, four cell populations emerge, phenotypically defined as CD34+CD41+ on day 7 (D7) and CD34+CD41+CD9- on D10 and D14 - qualified as "productive" because they can differentiate into proplatelet-forming cells during the D14-D17 period - and CD34-CD41+ or CD34+CD41+CD9+ on day 10 - qualified as "unproductive" because they are unable to form proplatelets later. Coculture with mesenchymal stem cells, or the presence of the AHR antagonist SR1, boosts the productive pathway in two ways: firstly, it increases the yield of D10 and D14 CD34+CD41+CD9- cells and secondly, it greatly increases their ability to generate proplatelets; in contrast, SR1 has no noticeable effect on the unproductive cell types. A transcriptome analysis was performed to decipher the genetic basis of these properties. This work represents the first extensive description of the genetic perturbations which accompany the differentiation of CD34+ progenitors into mature MKs at a subpopulation level. It highlights a wide variety of biological changes modulated in a time-dependent manner and allows anyone, according to his/her interests, to focus on specific biological processes accompanying MK differentiation. For example, the modulation of the expression of genes associated with cell proliferation, lipid and cholesterol synthesis, extracellular matrix components, intercellular interacting receptors and MK and platelet functions reflected the chronological development of the productive cells and pointed to unsuspected pathways. Surprisingly, SR1 only affected the gene expression profile of D10 CD34+CD41+CD9- cells; thus, as compared to these cells and those present on D14, the poorly productive D10 CD34+CD41+CD9- cells obtained in the absence of SR1 and the two unproductive populations present on D10 displayed an intermediate gene expression pattern. In other words, the ability to generate proplatelets between D10 and D14 appeared to be linked to the capacity of SR1 to delay MK differentiation, meanwhile avoiding intermediate and inappropriate genetic perturbations. Paradoxically, the D14 CD34+CD41+CD9- cells obtained under SR1- or SR1+ conditions were virtually identical, raising the question as to whether their strong differences in terms of proplatelet production, in the absence of SR1 and between D14 and D17, are mediated by miRNAs or by memory post-translational regulatory mechanisms.

cell biology↗