Mesenchymal stem/stromal cells (MSC) remain a promising tool for regenerative medicine as the efficacy of MSC-based cell therapy has been demonstrated for a broad spectrum of indications. Their therapeutic potency is mainly associated with their ability to secrete multiple factors critical for tissue regeneration. Due to comparable effects along with superior safety MSC conditioned medium (MSC-CM) containing a complex of MSC-secreted products is considered a reasonable alternative to cell therapy. However, the lack of standards regulating bioprocessing, use of proper auxiliary materials, and quality control complicates the development of MSC secretome-based therapeutics. In this study, we suggested several approaches addressing these issues. We manufactured 36 MSC-CM samples based on different xeno-free serum-free chemically defined media (DMEM-LG or MSC NutriStem^® XF) using original protocols and considered total concentrations of regeneration-associated paracrine factors secreted by human adipose-derived MSC at each time-point of conditioning. Using regression analysis, we retrospectively predicted associations between concentrations of several components of MSC-CM and its biological activity to stimulate human dermal fibroblast and endothelial cell migration in vitro as routine examples of potency assays for cell-based products. We also demonstrated that the cell culture medium might affect MSC-CM biological activity to varying degrees depending on the potency assay type. Furthermore, we showed that regression analysis might help to overcome donor variability. The suggested approaches might be successfully applied for other cell types if their secretome was shown to be promising for application in regenerative medicine.

We report a comparative study of multipotent mesenchymal stromal cells (MSC) delivered by injection, MSC-based cell sheets (CS) or MSC secretome to induce healing of cutaneous pressure ulcer in C57Bl/6 mice. We found that transplantation of CS from adipose-derived MSC resulted in reduction of fibrosis and recovery of skin structure with its appendages (hair and cutaneous glands). Despite short retention of CS on ulcer surface (3–7 days) it induced profound changes in granulation tissue (GT) structure, increasing its thickness and altering vascularization pattern with reduced blood vessel density and increased maturation of blood vessels. Comparable effects on GT vascularization were induced by MSC secretome, yet this treatment has failed to induce repair of skin with its appendages we observed in the CS group. Study of secretome components produced by MSC in monolayer or sheets revealed that CS produce more factors involved in pericyte chemotaxis and blood vessel maturation (PDGF-BB, HGF, G-CSF) but not sprouting inducer (VEGF165). Analysis of transcriptome using RNA sequencing and Gene Ontology mapping found in CS upregulation of proteins responsible for collagen binding and GT maturation as well as fatty acid metabolism enzymes known to be negative regulators of blood vessel sprouting. At the same time, downregulated transcripts were enriched by factors activating capillary growth, suggesting that in MSC sheets paracrine activity may shift towards matrix remodeling and maturation of vasculature, but not activation of blood vessel sprouting. We proposed a putative paracrine trigger mechanism potentially rendering an impact on GT vascularization and remodeling. Our results suggest that within sheets, MSC may change their functional state and spectrum of soluble factors that influence tissue repair and induce more effective skin healing inclining towards regeneration and reduced scarring.

Regeneration is a fundamental process attributed to the functions of adult stem cells. In the last decades, delivery of suspended adult stem cells is widely adopted in regenerative medicine as a leading means of cell therapy. However, adult stem cells cannot complete the task of human body regeneration effectively by themselves as far as they need a receptive microenvironment (the niche) to engraft and perform properly. Understanding the mechanisms underlying mammalian regeneration leads us to an assumption that improved outcomes of cell therapy require a specific microenvironment that is generated in damaged areas prior to stem cell delivery. To a certain extent, it may be achieved by the delivery of mesenchymal stromal cells (MSCs), not in dispersed form, but rather in self-organized cell sheets (CS) – tissue-like structures comprised of viable cells and microenvironment components: extracellular matrix and soluble factors deposited in the matrix. In this review, we highlight the potential role of MSCs as regeneration organizers and speculate that this function emerges in CS. This concept shifts our understanding of the therapeutic mechanism underlying a widely known CS-based delivery method for regenerative medicine.

Background: Injury of stem cell niches may disturb tissue homeostasis and regeneration coordinated by specific niche components. Yet, the mechanisms of stem cell niche restoration remain poorly understood. Herein, we examined the role of mesenchymal stromal cells (MSCs) as pivotal regulators of stem cell niche recovery focusing on the effects of their secretome. Methods: The spermatogonial stem cell (SSC) niche was selected as a model. SSC niches were injured by inducing abdominal cryptorchidism in rats. Briefly, testes of anesthetized rats were elevated into the abdominal cavity through the inguinal canal for 14 days. After descent of testes, MSC or MSC secretome treatment was applied to the animals by local subtunical injections. Results: Local administration of MSC or MSC secretome was sufficient to recover spermatogenesis and production of functional germ cells. The effects of MSC and their secreted components were comparable, leading to restoration of Sertoli cell pools and recovery of Leydig cell secretory functions. Conclusion: Our data suggest that MSCs mimic the functions of lost supportive cells within the stem cell niche, transiently providing paracrine stimuli for target cells and triggering tissue regenerative processes after damage.

The potential rapid advance of regenerative medicine was obstructed by findings that stimulation of human body regeneration is a much tougher mission than expected after the first cultures of stem and progenitor cells were established. In this mini review, we focus on the ambiguous role of growth factors in regeneration, discuss their evolutionary importance, and highlight them as the “cure and the cause” for successful or failed attempts to drive human body regeneration. We draw the reader's attention to evolutionary changes that occurred in growth factors and their receptor tyrosine kinases (RTKs) and how they established and shaped response to injury in metazoans. Discussing the well-known pleiotropy of growth factors, we propose an evolutionary rationale for their functioning in this specific way and focus on growth factors and RTKs as an amazing system that defines the multicellular nature of animals and highlight their participation in regeneration. We pinpoint potential bottlenecks in their application for human tissue regeneration and show their role in fibrosis/regeneration balance. This communication invites the reader to re-evaluate the functions of growth factors as keepers of natively existing communications between elements of tissue, which makes them a fundamental component of a successful regenerative strategy. Finally, we draw attention to the epigenetic landscape that may facilitate or block regeneration and give a brief insight into how it may define the outcome of injury.