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Mesenchymal stromal/stem cells (MSC) play a crucial role in promoting neovascularization, which is essential for wound healing. They are commonly utilized as an autologous source of progenitor cells in various stem cell-based therapies. However, incomplete MSC differentiation towards a vascular endothelial cell phenotype questions their involvement in an alternative process to angiogenesis, namely vasculogenic mimicry (VM), and the signal transducing events that regulate their in vitro priming into capillary-like structures. Here, human MSC were primed on top of Cultrex matrix to recapitulate an in vitro phenotype of VM. Total RNA was extracted, and differential gene expression assessed through RNA-Seq analysis and RT-qPCR. Transient gene silencing was achieved using specific siRNA. AG490, Tofacitinib, and PP2 pharmacological effects on VM structures were analyzed using the Wimasis software. In vitro VM occurred within 4 h and was prevented by the JAK/STAT3 inhibitors AG490 and Tofacitinib, as well as by the Src inhibitor PP2. RNA-Seq highlighted STAT3 as a signaling hub contributing to VM when transcripts from capillary-like structures were compared to those from cell monolayers. Concomitant increases in IL6, IL1b, CSF1, CSF2, STAT3, FOXC2, RPSA, FN1, and SNAI1 transcript levels suggest the acquisition of a combined angiogenic, inflammatory and epithelial-to-mesenchymal transition phenotype in VM cultures. Increases in STAT3, FOXC2, RPSA, Fibronectin, and Snail protein expression were confirmed during VM. STAT3 and RPSA gene silencing abrogated in vitro VM. In conclusion, in vitro priming of MSC into VM structures requires Src/JAK/STAT3 signaling. This molecular evidence indicates that a clinically viable MSC-mediated pseudo-vasculature process could temporarily support grafts through VM, allowing time for the host vasculature to infiltrate and remodel the injured tissues.

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Advanced bioinformatics analysis, such as systems biology (SysBio) and artificial intelligence (AI) approaches, including machine learning (ML) and deep learning (DL), is increasingly present in stem cell (SC) research. An approximate timeline on these developments and their global impact is still lacking. We conducted a scoping review on the contribution of SysBio and AI analysis to SC research and therapy development based on literature published in PubMed between 2000 and 2024. We identified an 8-10-fold increase in research output related to all three search terms between 2000 and 2021, with a 10-fold increase in AI-related production since 2010. Use of SysBio and AI still predominates in preclinical basic research with increasing use in clinically oriented translational medicine since 2010. SysBio- and AI-related research was found all over the globe, with SysBio output led by the United States (US, n=1487), United Kingdom (UK, n=1094), Germany (n=355), The Netherlands (n=339), Russia (n=215), and France (n=149), while for AI-related research the US (n=853) and UK (n=258) take a strong lead, followed by Switzerland (n=69), The Netherlands (n=37), and Germany (n=19). The US and UK are most active in SCs publications related to AI/ML and AI/DL. The prominent use of SysBio in ESC research was recently overtaken by prominent use of AI in iPSC and MSC research. This study reveals the global evolution and growing intersection between AI, SysBio, and SC research over the past two decades, with substantial growth in all three fields and exponential increases in AI-related research in the past decade.

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Platelet lysate (PL) is investigated as a potential replacement for fetal bovine serum (FBS) in cell culture. However, there is limited research on its impact on the immune profile of equine mesenchymal stromal cells (eMSCs). This study aimed to evaluate the effects of different PL formulations on the proliferative capacity, multipotentiality, and immune profile of equine adipose tissue-derived MSCs (eAD-MSCs). In vitro growth kinetics and trilineage differentiation of eAD-MSCs (n = 7) were assessed under three culture conditions: medium-concentration PL (MPL), high-concentration PL (HPL), and FBS as a control. The immune profile was evaluated by studying the expression of immunogenic receptors such as MHC I, MHC II, and immunomodulatory molecules IL-6, IL-10, and TNF-α, determined by gene expression, surface marker expression, and cytokine quantification. Both PL formulations, pooled from 5 donors, exhibited 3.3 and 6.5-fold higher platelet counts than baseline plasma for MPL and HPL, respectively. Higher concentrations of TGF-ß and PDGF were found in both PL formulations compared to baseline. Furthermore, MPL and HPL subcultures demonstrated proliferative, clonogenic, and multipotent capacities similar to FBS. The immune profile of PL-cultured cells exhibited gene expression levels related to immunogenicity and immunomodulation similar to the reference condition, and the surface antigen presence of MHC II was also similar. However, HPL media exhibited higher IL-6, IL-10, and TNF-α concentrations in the culture supernatant. In conclusion, both PL media contained higher concentrations of growth factors compared to FBS, supporting the in vitro culture of eAD-MSCs with proliferative, clonogenic, and multipotent capacity similar to the reference medium. Nonetheless, PL usage led to a variation in the immunomodulatory cytokine microenvironment, with higher concentrations of IL-6, IL-10, and TNF-α in HPL media compared to MPL and FBS.

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BACKGROUND: Stromal Vascular fraction/gel (SVF/gel) is prepared mechanically from autologous adipose tissue, and it is known for its regenerative and anti-inflammatory properties. AIMS: To assess histopathological effects of adipose tissue-derived SVF/gel and nasal steroids on nasal mucosal healing. MATERIAL AND METHODS: Forty-two Wistar Albino rats with right nasal mucosal injury were randomly divided into three groups: control (saline), Mometasone Furoate (MF), and SVF/gel. Control group (n = 14) received saline for 7 days, while MF group (n = 14) was administered MF to the right nasal cavity for 7 days. SVF/gel group (n = 14) was treated once with SVF/gel in the right nasal cavity. Histological analysis on days 14 and 28 post-injury focused on evaluating epithelial thickness, inflammation, disarray, subepithelial thickness, goblet cell count, subepithelial fibrosis, presence of ciliated cells, lacunae, adhesion, and neo-osteogenesis. RESULTS: When comparing the MF and SVF/gel groups, statistically significant differences were found on day 14 in indices of epithelial thickness, subepithelial thickness, goblet cells, subepithelial fibrosis, and ciliated cells. On day 28, SVF/gel group exhibited higher ciliated cell counts and lower subepithelial fibrosis values (p = .027; p = .016). Additionally, epithelial disarray, adhesions, lacunae, and neo-osteogenesis were not observed in the SVF/gel group. CONCLUSIONS AND SIGNIFICANCE: SVF/gel accelerates re-epithelialization, reduces fibrosis and adhesions, and enhances cilia formation compared to nasal steroids. These findings suggest that SVF/gel is an autologous and cost-effective treatment for improving nasal mucosal healing post-injury.

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BACKGROUND: Knee osteoarthritis (KOA) presents a prevalent orthopedic condition causing substantial impairment in the quality of life and imposing a significant societal and economic burden. Mesenchymal stromal/stem cells (MSCs), known for their regenerative properties and immunomodulatory effects, have emerged as a promising therapeutic avenue in regenerative medicine. Despite MSCs' therapeutic potential, their precise mechanisms of action in KOA remain underexplored. METHODS: Conducted as a randomized, open-label clinical trial, 20 patients will be enrolled, with 10 in the intervention group and 10 in the control group. The primary focus will be to explore the molecular mechanisms associated with MSC therapy. Biomarkers and gene expressions related to cartilage metabolism, inflammation, immune modulation, and pain in the synovial fluid, blood, and tissue samples will be analyzed. Patients will undergo pre- and post-treatment evaluations using patient-reported outcome measures (PROMs) and comprehensive clinical assessments. DISCUSSION: This is an exploratory study with the goal to provide comprehensive insights into the therapeutic effects of MSCs on a molecular level, potentially paving the way for optimized and more effective MSC-based therapies in the management of KOA, as well as furthering the development of novel treatment strategies. TRIAL REGISTRATION: ClinicalTrials.gov, NCT06078059. Registered on 5 October 2023.

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Mesenchymal stromal/stem cells (MSCs) and their secretome are known to exert beneficial effects in many pathological states. However, MSCs therapeutic properties can be reduced due to unsuitable in vitro maintenance conditions. Standard culture protocols neglect the fact that MSCs exist in vivo in the closest connection with the extracellular matrix (ECM), the complex protein network providing an instructive microenvironment. We found recently that conditioned medium from human endometrial MSCs cultured on cell-derived decellularized extracellular matrix (CM-dECM) is dramatically enriched in a number of paracrine factors such as GM-CSF, FGF-2, HGF, MMP-1, MCP-1, IL-6, IL-8, CXCL-1, -2, -5, -6 (Ushakov et al., 2024). Given that several upregulated molecules belong to myokines that are known to participate in skeletal muscle regeneration, we hypothesized that CM-dECM may promote restoration of damaged muscle tissue. Here, we found that CM-dECM injections into barium chloride-injured murine m. tibialis anterior caused myofiber hypertrophy and promoted angiogenesis. Besides, CM-dECM significantly contributed to progression of murine C2C12 myoblasts cell cycle suggesting that muscle repair in vivo may be connected with stimulation of resident myoblasts proliferation. In this study, a role for secretome of endometrial MSCs cultured on dECM in injured murine skeletal muscle regeneration was outlined first. Our findings demonstrate that culture on dECM may be considered as a novel preconditioning approach enhancing MSCs therapeutic potential.

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BACKGROUND: AT-MSCs display great immunoregulatory features, making them potential candidates for cell-based therapy. This study aimed to evaluate the "RBC lysis buffer" isolation protocol and immunological profiling of the so-obtained AT-MSCs. METHODS: We established an immune-comparative screening of AT-MSCs throughout in vitro cell expansion (PM, P1, P2, P3, P4) and inflammatory priming regarding the expression of 28 cell-surface markers, 6 cytokines/chemokines, and 10 TLR patterns. FINDINGS: AT-MSCs were highly expandable and sensitive to microenvironment challenges, hereby showing plasticity in distinct expression profiles. Both cell expansion and inflammation differentially modulated the expression profile of CD34, HLA-DR, CD40, CD62L, CD200 and CD155, CD252, CD54, CD58, CD106, CD274 and CD112. Inflammation resulted in a significant increase in the expression of the cytokines IL-6, IL-8, IL-1ß, IL-1Ra, CCL5, and TNFα. Depending on the culture conditions, the expression of the TLR pattern was distinctively altered with TLR1-4, TLR7, and TLR10 being increased, whereas TLR6 was downregulated. Protein network and functional enrichment analysis showed that several trophic and immune responses are likely linked to these immunological changes. CONCLUSIONS: AT-MSCs may sense and actively respond to tissue challenges by modulating distinct and specific pathways to create an appropriate immuno-reparative environment. These mechanisms need to be further characterized to identify and assess a molecular target that can enhance or impede the therapeutic ability of AT-MSCs, which therefore will help improve the quality, safety, and efficacy of the therapeutic strategy.

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This chapter introduces the increasing significance of mesenchymal stromal/stem cell (MSC) production in regenerative medicine and cellular therapeutics, outlines the growing interest in MSCs for various medical applications, and highlights their potential in advanced therapy medicinal products (ATMPs) and the advancements in cell culture technologies that have facilitated large-scale MSC production under Good Manufacturing Practices (GMP), ensuring safety and efficacy. This chapter describes an optimized upstream protocol for laboratory-scale MSC production from different tissue sources. This protocol, conducted in flasks, controls critical parameters and lays the foundation for downstream processing to generate ATMPs. This comprehensive approach underscores the potential of MSCs in clinical applications and the importance of tailored production processes.

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Mesenchymal stromal cells (MSCs) are investigated as cellular therapeutics for inflammatory bowel diseases and associated perianal fistula, although consistent efficacy remains a concern. Determining host factors that modulate MSCs' potency including their secretion of angiogenic and wound-healing factors, immunosuppression, and anti-inflammatory properties are important determinants of their functionality. We investigated the mechanisms that regulate the secretion of angiogenic and wound-healing factors and immune suppression of human bone marrow MSCs. Secretory analysis of MSCs focusing on 18 angiogenic and wound-healing secretory molecules identified the most abundancy of vascular endothelial growth factor A (VEGF-A). MSC viability and secretion of other angiogenic factors are not dependent on VEGF-A secretion which exclude the autocrine role of VEGF-A on MSC's fitness. However, the combination of inflammatory cytokines IFNγ and TNFα reduces MSC's VEGF-A secretion. To identify the effect of intestinal microvasculature on MSCs' potency, coculture analysis was performed between human large intestine microvascular endothelial cells (HLMVECs) and human bone marrow-derived MSCs. HLMVECs do not attenuate MSCs' viability despite blocking their VEGF-A secretion. In addition, HLMVECs neither attenuate MSC's IFNγ mediated upregulation of immunosuppressive enzyme indoleamine 2,3-dioxygenase nor abrogate suppression of T-cell proliferation despite the attenuation of VEGF-A secretion. We found that HLMVECs express copious amounts of endothelial nitric oxide synthase and mechanistic analysis showed that pharmacological blocking reverses HLMVEC-mediated attenuation of MSC's VEGF-A secretion. Together these results suggest that secretion of VEGF-A and immunosuppression are separable functions of MSCs which are regulated by distinct mechanisms in the host.

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Mesenchymal stromal/stem cells (MSCs) are a heterogeneous population of multipotent cells that can be obtained from various tissues, such as dental pulp, adipose tissue, bone marrow and placenta. MSCs have gained importance in the field of regenerative medicine because of their promising role in cell therapy and their regulatory abilities in tissue repair and regeneration. However, a better characterization of these cells and their products is necessary to further potentiate their clinical application. In this study, we used unbiased high-resolution mass spectrometry-based proteomic analysis to investigate the impact of distinct priming strategies, such as hypoxia and IFN-γ treatment, on the composition and therapeutic functionality of the secretome produced by MSCs derived from the amniotic membrane of the human placenta (hAMSCs). Our investigation revealed that both types of priming improved the therapeutic efficacy of hAMSCs, and these improvements were related to the secretion of functional factors present in the conditioned medium (CM) and exosomes (EXOs), which play crucial roles in mediating the paracrine effects of MSCs. In particular, hypoxia was able to induce a pro-angiogenic, innate immune response-activating, and tissue-regenerative hAMSC phenotype, as highlighted by the elevated production of regulatory factors such as VEGFA, PDGFRB, ANGPTL4, ENG, GRO-γ, IL8, and GRO-α. IFN-γ priming, instead, led to an immunosuppressive profile in hAMSCs, as indicated by increased levels of TGFB1, ANXA1, THBS1, HOMER2, GRN, TOLLIP and MCP-1. Functional assays validated the increased angiogenic properties of hypoxic hAMSCs and the enhanced immunosuppressive activity of IFN-γ-treated hAMSCs. This study extends beyond the direct priming effects on hAMSCs, demonstrating that hypoxia and IFN-γ can influence the functional characteristics of hAMSC-derived secretomes, which, in turn, orchestrate the production of functional factors by peripheral blood cells. This research provides valuable insights into the optimization of MSC-based therapies by systematically assessing and comparing the priming type-specific functional features of hAMSCs. These findings highlight new strategies for enhancing the therapeutic efficacy of MSCs, particularly in the context of multifactorial diseases, paving the way for the use of hAMSC-derived products in clinical practice.

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Intercellular mitochondrial transfer (MT) is a newly discovered form of cell-to-cell signalling involving the active incorporation of healthy mitochondria into stressed/injured recipient cells, contributing to the restoration of bioenergetic profile and cell viability, reduction of inflammatory processes and normalisation of calcium dynamics. Recent evidence has shown that MT can occur through multiple cellular structures and mechanisms: tunneling nanotubes (TNTs), via gap junctions (GJs), mediated by extracellular vesicles (EVs) and other mechanisms (cell fusion, mitochondrial extrusion and migrasome-mediated mitocytosis) and in different contexts, such as under physiological (tissue homeostasis and stemness maintenance) and pathological conditions (hypoxia, inflammation and cancer). As Mesenchimal Stromal/ Stem Cells (MSC)-mediated MT has emerged as a critical regulatory and restorative mechanism for cell and tissue regeneration and damage repair in recent years, its potential in stem cell therapy has received increasing attention. In particular, the potential therapeutic role of MSCs has been reported in several articles, suggesting that MSCs can enhance tissue repair after injury via MT and membrane vesicle release. For these reasons, in this review, we will discuss the different mechanisms of MSCs-mediated MT and therapeutic effects on different diseases such as neuronal, ischaemic, vascular and pulmonary diseases. Therefore, understanding the molecular and cellular mechanisms of MT and demonstrating its efficacy could be an important milestone that lays the foundation for future clinical trials.

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BACKGROUND: The impact of global overconsumption of simple sugars on bone health, which peaks in adolescence/early adulthood and correlates with osteoporosis (OP) and fracture risk decades, is unclear. Mesenchymal stromal/stem cells (MSCs) are the progenitors of osteoblasts/bone-forming cells, and known to decrease their osteogenic differentiation capacity with age. Alarmingly, while there is correlative evidence that adolescents consuming greatest amounts of simple sugars have the lowest bone mass, there is no mechanistic understanding on the causality of this correlation. METHODS: Bioinformatics analyses for energetics pathways involved during MSC differentiation using human cell information was performed. In vitro dissection of normal versus high glucose (HG) conditions on osteo-/adipo-lineage commitment and mitochondrial function was assessed using multi-sources of non-senescent human and murine MSCs; for in vivo validation, young mice was fed normal or HG-added water with subsequent analyses of bone marrow CD45- MSCs. RESULTS: Bioinformatics analyses revealed mitochondrial and glucose-related metabolic pathways as integral to MSC osteo-/adipo-lineage commitment. Functionally, in vitro HG alone without differentiation induction decreased both MSC mitochondrial activity and osteogenesis while enhancing adipogenesis by 8 h' time due to depletion of nicotinamide adenine dinucleotide (NAD+), a vital mitochondrial co-enzyme and co-factor to Sirtuin (SIRT) 1, a longevity gene also involved in osteogenesis. In vivo, HG intake in young mice depleted MSC NAD+, with oral NAD+ precursor supplementation rapidly reversing both mitochondrial decline and osteo-/adipo-commitment in a SIRT1-dependent fashion within 1 ~ 5 days. CONCLUSIONS: We found a surprisingly rapid impact of excessive glucose, a single dietary factor, on MSC SIRT1 function and osteogenesis in youthful settings, and the crucial role of NAD+-a single molecule-on both MSC mitochondrial function and lineage commitment. These findings have strong implications on future global OP and disability risks in light of current worldwide overconsumption of simple sugars.

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BACKGROUND AIMS: Cytopenias after allogeneic stem cell transplantation (allo-SCT) are a common complication, the underlying pathogenic mechanisms of which remain incompletely understood. Multipotent mesenchymal stromal/stem cell (MSC) therapy has been successfully employed in the treatment of immune-related disorders and can aid in the restoration of the hematopoietic niche. METHODS: A phase II clinical trial to assess the efficacy and safety of administering four sequential doses of ex-vivo expanded bone marrow MSCs from a third-party donor to patients with persistent severe cytopenias after allo-SCT was performed. RESULTS: The overall response rate on day 90 was 75% among the 27 evaluable patients (comprising 12 complete responses, 8 partial responses, and 7 with no response). The median time to respond was 14.5 days. Responses were observed across different profiles, including single or multiple affected lineages, primary or secondary timing, and potential immune-mediated or post-infectious pathophysiology versus idiopathic origin. With a median follow-up for surviving patients of 85 months after MSC infusion, 53% of patients are alive. Notably, no adverse events related to MSC therapy were reported. CONCLUSIONS: In summary, the sequential infusion of third-party MSCs emerges as a viable and safe therapeutic option, exhibiting potential benefits for patients experiencing cytopenias following allo-SCT.

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Mesenchymal stromal/stem cells (MSCs), which are distributed in many tissues including bone marrow, have been reported to play a critical role in tumor development. While bone marrow, the primary site for hematopoiesis, is important for establishing the immune system, whether MSCs in the bone marrow can promote tumor growth via influencing hematopoiesis remains unclear. We observed that the numbers of MSCs and neutrophils were increased in bone marrow in tumor-bearing mice. Moreover, co-culture assay showed that MSCs strongly protected neutrophils from apoptosis and induced their maturation. G-CSF and GM-CSF have been well-documented to be associated with neutrophil formation. We found a remarkably increased level of G-CSF, but not GM-CSF, in the supernatant of MSCs and the serum of tumor-bearing mice. The G-CSF expression can be enhanced with inflammatory cytokines (IFNγ and TNFα) stimulation. Furthermore, we found that IFNγ and TNFα-treated MSCs enhanced their capability of promoting neutrophil survival and maturation. Our results indicate that MSCs display robustly protective effects on neutrophils to contribute to tumor growth in bone niches.

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Residual substances that are considered hazardous to the recipient must be removed from final cellular therapeutic products manufactured for clinical purposes. In doing so, quality rules determined by competent authorities (CAs) for the clinical use of tissue- and cell-based products can be met. In our study, we carried out residual substance analyses, and purity determination studies of trypsin and trypsin inhibitor in clinically manufactured bone marrow-derived mesenchymal stromal/stem cell products, using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method. Despite being a semiquantitative method, SDS-PAGE has several benefits over other methods for protein analysis, such as simplicity, convenience of use, and affordability. Due to its convenience and adaptability, SDS-PAGE is still a commonly used method in many laboratories, despite its limits in dynamic range and quantitative precision. Our goal in this work was to show that SDS-PAGE may be used effectively for protein measurement, especially where practicality and affordability are the major factors. The results of our study suggest a validated method to guide tissue and cell manufacturing sites for making use of an agreeable, accessible, and cost-effective method for residual substance analyses in clinically manufactured cellular therapies.

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Adipose tissue is an abundant and accessible source of stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. Adipose-derived stromal/stem cells (ASCs) have been widely used in tissue engineering and cell therapy. In addition, the clinical application of ASCs in the treatment of inflammation and injury has been proven a success. Here, we describe methods from our own laboratory and the literature for the isolation and expansion of Adipose-derived stromal/stem cells (ASCs). We present a large-scale procedure suitable for processing >100 mL volumes of lipoaspirate tissue specimens by collagenase digestion, a related procedure suitable for processing adipose tissue aspirates without digestion, and a procedure suitable for intact human adipose tissue, such as buccal fat pads in the maxillofacial region.

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Adipose tissue provides a valuable cell source for tissue engineering, regenerative medicine, and adipose tissue biology studies. The most widely used adipose-derived stromal/stem cells (ASCs) isolation protocol involves enzymatic digestion with collagenase. However, the yield of the method often proves to be poor if not impossible for collection of sufficient stromal vascular fraction (SVF) for expansion when the sample size is small, for instance when only newborn mice are available for cell culture. Here, we describe an efficient protocol for the isolation and expansion of ASCs using explant culture as an alternative. Briefly, adipose tissue was minced after removing excess liquid. Then, the minced tissue was placed in culture dishes or flasks. The cells will migrate out of tissue and adhere to the culture surface after one or more days.

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The tumor microenvironment is importantly shaped by various cytokines, where interleukins (ILs) and interferons (IFNs) shape the balance of immune activity within tumor niche and associated lymphoid organs. Their importance in activation and tuning of both innate and adaptive immune responses prompted their use in several clinical trials, albeit with limited therapeutic efficacy and risk of toxicity due to systemic administration. Increasing preclinical evidence suggests that local delivery of ILs and IFNs could significantly increase their effectiveness, while simultaneously attenuate the known side effects and issues related to their biological activity. A prominent way to achieve this is to use cell-based delivery vehicles. For this purpose, mesenchymal stromal stem cells (MSCs) are considered an almost ideal candidate. Namely, MSCs can be obtained in large quantities and from obtainable sources (e.g. umbilical cord or adipose tissue), their ex vivo expansion is relatively straightforward compared to other cell types and they possess very low immunogenicity making them suitable for allogeneic use. Importantly, MSCs have shown an intrinsic capacity to respond to tumor-directed chemotaxis. This review provides a focused and detailed discussion on MSC-based gene therapy using ILs and IFNs, engineering techniques and insights on potential future advancements.

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Alpha-1 antitrypsin-overexpressing mesenchymal stromal/stem cells (AAT-MSCs) showed improved innate properties with a faster proliferation rate when studied for their protective effects in mouse models of diseases. Here, we investigated the potential mechanism(s) by which AAT gene insertion increases MSC proliferation. Human bone marrow-derived primary or immortalized MSCs (iMSCs) or AAT-MSCs (iAAT-MSCs) were used in the study. Cell proliferation was measured by cell counting and cell cycle analysis. Possible pathways involved in the pro-proliferation effect of AAT were investigated by measuring mRNA and protein expression of key cell cycle genes. Interval cell counting showed increased proliferation in AAT-MSCs or iAAT-MSCs compared to their corresponding MSC controls. Cell cycle analysis revealed more cells progressing into the S and G2/M phases in iAAT-MSCs, with a notable increase in the cell cycle protein, Cyclin D1. Moreover, treatment with Cyclin D1 inhibitors showed that the increase in proliferation is due to Cyclin D1 and that the AAT protein is upstream and a positive regulator of Cyclin D1. Furthermore, AAT's effect on Cyclin D1 is independent of the Wnt signaling pathway as there were no differences in the expression of regulatory proteins, including GSK3ß and ß-Catenin in iMSC and iAAT-MSCs. In summary, our results indicate that AAT gene insertion in an immortalized MSC cell line increases cell proliferation and growth by increasing Cyclin D1 expression and consequently causing cells to progress through the cell cycle at a significantly faster rate.

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