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Cytotherapy Corner May 2023


Donald G. Phinney, PhD 
Senior Editor Department of Molecular Medicine
UF Scripps Biomedical Research 
Jupiter, FL, USA

In this issue of Cytotherapy Corner, I want to highlight a manuscript in the April 2023 issue by Huang et al., that characterized soluble products released from necrotic and apoptotic mesenchymal stem cells (MSCs) and evaluated their potency in preventing muscle injury in an animal model of peripheral artery disease. The paper is timely since various studies have demonstrated the MSC secretome replicates in large part the therapeutic impacts of parental cells in pre-clinical disease models through various mechanisms including pro-inflammatory licensing, release of extracellular vesicles, apoptotic phenomenon including efferocytosis, and induction of an immediate blood mediated inflammatory response. In the current study, the authors produced apoptotic and necrotic MSCs by exposing them to three cycles of freeze/thaw (F&T; liquid nitrogen for 2 minutes followed by 4 minutes at 37°C) or heating cells a 55°C for 20 minutes, then isolated both the cell pellets and supernatant for downstream analysis. First, the authors quantified impacts of the processed components on macrophages, which revealed the F&T components exhibited an anti-inflammatory and pro-angiogenic response based on gene and protein expression profiling in macrophages. Importantly, the authors further demonstrated that impacts of the F&T cell pellet were ameliorated by pre-treatment with a phosphatidylserine (PtS) antibody. As a control, the authors also prepared F&T supernatant fractions from two mouse cell lines, NIH 3T3 and RAW 264.7, and found the MSC and 3T3 fractions were equally effective in reducing TNFα secretion from macrophages while that from  RAW cells was significantly less potent. They then conducted a series of studies demonstrating the active component of the F&T supernatant fraction was proteinaceous, and identified several candidates including PAI-I, M-CSF, and IL-33 as highly enriched in MSC and 3T3 but not RAW F&T supernatant fractions. Lastly, they administered F&T supernatant from MSCs to mice subjected to femoral artery ligation and evaluated impacts on muscle mass, tetanic force, and blood flow.  Herein, F&T supernatant treated muscles yielded a significantly higher average tetanic force compared to untreated muscle but no differences in muscle mass were found across the treatment groups, which also included naïve MSCs and supernatant from heat treated MSCs.

In addition, F&T supernatant treatment increased average myofiber size, decreased fibrosis, prevented deposition of neutral fat, and improved relative blood flow in the affected foot to ~80% of the intact foot. Lastly, the F&T supernatant treated muscle has significantly lower numbers of macrophages compared to all other treatment groups, indicative of accelerated resolution of inflammation. Overall, the study further advances knowledge and efforts aimed at exploiting apoptosis mechanisms to improve therapeutic outcomes of MSC-based therapies, which have been largely suboptimal despite widespread clinical testing.  

In the May Issue, I want to draw attention to a paper by Klein et al., from the Stem Cell Engineering Committee of ISCT that reviews critical parameters influencing the use of hematopoietic stem cell transplantation (HSCT) for patients with non-malignant disease. The topic is important since HSCT is curative for many non-malignant diseases, but outcomes are highly dependent on optimal disease management pre-treatment and choice of donor, conditioning, and post-transplant management.  For example, there is no benefit to graft vs. tumor responses in patients with non-malignant diseases, and while matched sibling donors are typically the best choice for patients with malignant disease, these donors may be carriers of the disease allele and therefore not eligible as donors for patients with non-malignant disease. Expertly written, the paper systematically reviews pre-transplant considerations, overall transplant goals, conditioning regimens, donor choice, and graft manipulation techniques that may be considered in developing an optimized HSCT program for patients with non-malignant disease. The authors also provide succinct summaries of the relevant variables/parameters that should be considered when developing transplant programs for these patients. These include efforts to reduce the toxicity of preparative regimens, expanding the donor pool, and enhancing immune reconstitution post-transplant with the ultimate goal of improving outcomes and expanding the range of applicable diseases that may benefit from HSCT. 

Happy reading.   

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