Background The R-Spondin proteins comprise a family of secreted proteins, known for their important roles in cell proliferation, differentiation and death, by inducing the Wnt pathway. Several studies have demonstrated the importance of RSPOs in regulation of a number of tissue-specific processes, namely: bone formation, skeletal muscle tissue development, proliferation of pancreatic β-cells and intestinal stem cells and even cancer. RSPO1 stands out among RSPOs molecules with respect to its potential therapeutic use, especially in the Regenerative Medicine field, due to its mitogenic activity in stem cells. Here, we generated a recombinant human RSPO1 (rhRSPO1) using the HEK293 cell line, obtaining a purified, characterized and biologically active protein product to be used in Cell Therapy. The hRSPO1 coding sequence was synthesized and subcloned into a mammalian cell expression vector. HEK293 cells were stably co-transfected with the recombinant expression vector containing the hRSPO1 coding sequence and a hygromycin resistance plasmid, selected for hygror and subjected to cell clones isolation. Results rhRSPO1 was obtained, in the absence of serum, from culture supernatants of transfected HEK293 cells and purified using a novel purification strategy, involving two sequential chromatographic steps, namely: heparin affinity chromatography, followed by a molecular exclusion chromatography, designed to yield a high purity product. The purified protein was characterized by Western blotting, mass spectrometry and in vitro (C2C12 cells) and in vivo (BALB/c mice) biological activity assays, confirming the structural integrity and biological efficacy of this human cell expression system. Furthermore, rhRSPO1 glycosylation analysis allowed us to describe, for the first time, the glycan composition of this oligosaccharide chain, confirming the presence of an N-glycosylation in residue Asn137 of the polypeptide chain, as previously described. In addition, this analysis revealing the presence of glycan structures such as terminal sialic acid, N-acetylglucosamine and/or galactose. Conclusion Therefore, a stable platform for the production and purification of recombinant hRSPO1 from HEK293 cells was generated, leading to the production of a purified, fully characterized and biologically active protein product to be applied in Tissue Engineering.

Mesenchymal stem cells (MSCs) are crucial for tissue homeostasis and repair, secreting vesicles to the extracellular environment. Isolated exosomes were shown to affect angiogenesis, immunomodulation and tissue regeneration. Numerous efforts have been dedicated to describe the mechanism of action of these extracellular vesicles (EVs) and guarantee their safety, since the final aim is their therapeutic application in the clinic. The major advantage of applying MSC-derived EVs is their low or inexistent immunogenicity, prompting their use as drug delivery or therapeutic agents, as well as wound healing, different cancer types, and inflammatory processes in the neurological and cardiovascular systems. MSC-derived EVs display no vascular obstruction effects or apparent adverse effects. Their nano-size ensures their passage through the blood–brain barrier, demonstrating no cytotoxic or immunogenic effects. Several in vitro tests have been conducted with EVs obtained from different sources to understand their biology, molecular content, signaling pathways, and mechanisms of action. Application of EVs to human therapies has recently become a reality, with clinical trials being conducted to treat Alzheimer’s disease, retina degeneration, and COVID-19 patients. Herein, we describe and compare the different extracellular vesicles isolation methods and therapeutic applications regarding the tissue repair and regeneration process, presenting the latest clinical trial reports.

Effect of recombinant human peptide growth factors (PDGF-BB and/or VEGF165) at mesenchymal stem cells secretoma and in a wound healing animal modelRegenerative Medicine aims to achieve treatments that accelerate the different stages of the healing process in both humans and animals, and the manipulation of the growth factor (GF) composition can improve or modify the repair and remodeling process of injured tissues, as well as providing more aesthetic and functional scars. The present study aimed to evaluate the effect of recombinant human platelet-derived growth factor (PDGF-BB) and/or endothelial vascular growth factor (VEGF165) on adipose (canine) and bone marrow (equine) mesenchymal stem cells and their secretome, as well as in a model of skin healing in nude rats. GF were obtained from purified media by heparin affinity chromatography and characterized by ELISA and Western blot, and were used in vehicle buffer or in 2% alginate hydrogel. Mesenchymal stem cells were obtained, characterized, cultured and treated with GF and exosomes were isolated from conditioned media. The animal model of wound healing was established in Rowett nude rats, where 3µg/mL of GF were applied alone or combined in wounds on the animals' backs, and after 7 days the healing evolution was evaluated macroscopically and histopathologically. The present study demonstrated for the first time the potential of rhPDGF-BB and/or rhVEGF165 supplementation in the healing of skin wounds in seven days, stimulating angiogenesis, fibroblast reaction and re-epithelialization, but a longterm evaluation is necessary. The 2% alginate hydrogel proved to be the best approach to be used as a vehicle for GF applications in vivo in relation to the vehicle buffer, but not in vitro assays. The treatment with both factors combined improved the therapeutic profile of adipose mesenchymal stem cells by stimulating cell migration and exosome secretion. The study demonstrated for the first time that treatment with rhVEGF165 stimulated cell proliferation, angiogenic profile and secretion of exosomes by equine bone marrow mesenchymal stem cells. Together, the data support future investigations into the effect of GF supplementation in vitro, on cellular and molecular content of

The rabbit is considered an ideal animal model for studies that describe abnormalities in the testicles due to the similar morphogenetic mechanisms of sexual development and diseases commonly found in humans. The aim of this study was to determine the male sexual differentiation of the New Zealand rabbit (Oryctolagus cuniculus) through development. The gestational age was estimated and classified as 9, 12, 14, 16, 18, 20, 23 and 28 gestational days. The morphological and sexual determination were performed by histological analysis of the reproductive tract in the embryos and fetuses (9-28 days) as well as by immunohistochemistry-Desert hedgehog-Dhh-(testis-specific protein on Y chromosome-16, 20, 23 days and adult rabbits). Gonads were observed from the 14 th day in an undifferentiated stage and with homogeneous aspect. Sexual differentiation was observed from the 16 th day with presence of cells forming gonadal cords and Dhh + cells in the gonadal parenchyma. From the 18 th gestational day testicular cords were observed, which evolved into organized seminiferous tubules. The formation of the efferent ducts and ductus deferens and epididymis was observed on the 20 th and 23 rd days, respectively. The differentiation of the external genitalia occurred from the 23 rd days from the anogenital distance and was identified to identify the penile structures. In summary, the features of the sexual differentiation were determined by observation of the Dhh + protein in embryos from the 16 th day to adulthood, and the morphological particularities observed from the 18 th gestational day, determined by differentiation of the external genitalia from the 23 rd day.