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Mesenchymal stromal cells (MSCs) have recently emerged as an interesting therapeutic approach for patients with progressive systemic sclerosis (SSc), a rare and life-threatening orphan autoimmune disease. Whereas MSC immunomodulatory potential is considered as a central mechanism for their clinical benefit, very few data are available on the impact of MSCs on immune cell subsets in vivo. In the current extended study of a phase I/II clinical trial exploring the injection of a single dose of allogeneic bone marrow-MSCs (alloBM-MSCs) in patients with severe SSc (NCT02213705), we performed a longitudinal in-depth characterization of circulating immune cells in 19 MSC-treated patients, including 14 responders and 5 non-responders. By a combination of flow cytometry and transcriptomic analyses, we highlighted an increase in circulating CD24hiCD27posCD38lo/neg memory B cells, the main IL-10-producing regulatory B cell (Breg) subset, and an upregulation of IL10 expression in ex-vivo purified B cells, specifically in responder patients, early after the alloBM-MSC infusion. In addition, a deeper alteration of the B-cell compartment before alloBM-MSC treatment, including a higher expression of profibrotic cytokines IL6 and TGFβ by sorted B cells was associated with a non-responder clinical status. Finally, BM-MSCs were able to directly upregulate IL-10 production in activated B cells in vitro. These data suggest that cytokine-producing B cells, in particular Breg, are pivotal effectors of BM-MSC therapeutic activity in SSc. Their quantification as activity biomarkers in MSC potency assays and patient selection criteria may be considered to reach optimal clinical benefit when designing MSC-based clinical trials.

Calcium phosphates (CaP) are widely used synthetic bone graft substitutes, having bioactivity that is regulated by a set of intertwined physico-chemical and structural properties. While some CaPs have shown to be as effective in regenerating large bone defects as autologous bone, there is still the need to understand the role of individual material properties in CaP performance. Here, we aimed to decouple the effects of chemical composition and surface-microstructure of a beta-tricalcium phosphate (TCP) ceramic, with proven osteoinductive potential, on human mesenchymal stromal cells (hMSCs) differentiation. To this end, we replicated the surface structure of the TCP ceramic into polylactic acid without inorganic additives, or containing the chemical constituents of the ceramic, i.e., a calcium salt, a phosphate salt, or TCP powder. The microstructure of the different materials was characterized by confocal laser profilometry. hMSCs were cultured on the materials, and the expression of a set of osteogenic genes was determined. The cell culture medium was collected and the levels of calcium and phosphate ions were quantified by inductively-coupled plasma mass spectrometry. The results revealed that none of the tested combinations of properties in polymer/composite replicas was as potent in supporting the osteogenic differentiation of hMSCs as the original ceramic. Nevertheless, we observed some effects of the surface structure in the absence of inorganics, as well as combined effects of surface structure and the added salts, in particular calcium, on osteogenic differentiation. The approach presented here can be used to study the role of independent properties in other CaP-based biomaterials.

Quantitative polymerase chain reaction (qPCR) has emerged as an important bioanalytical method for assessing the pharmacokinetics of human-cell-based medicinal products after xenotransplantation into immunodeficient mice. A particular challenge in bioanalytical qPCR studies is that the different tissues of the host organism can affect amplification efficiency and amplicon detection to varying degrees, and ignoring these matrix effects can easily cause a significant underestimation of the true number of target cells in a sample. Here, we describe the development and drug regulatory-compliant validation of a TaqMan® qPCR assay for the quantification of mesenchymal stromal cells in the range of 125 to 20,000 cells/200 µL lysate via the amplification of a human-specific, highly repetitive α-satellite DNA sequence of the chromosome 17 centromere region HSSATA17. An assessment of matrix effects in 14 different mouse tissues and blood revealed a wide range of spike recovery rates across the different tissue types, from 11 to 174%. Based on these observations, we propose performing systematic spike-and-recovery experiments during assay validation and correcting for the effects of the different tissue matrices on cell quantification in subsequent bioanalytical studies by multiplying the back-calculated cell number by tissue-specific factors derived from the inverse of the validated percent recovery rate.

We evaluated a dimethyl sulfoxide (Me2SO)-free cryopreservation solution to freeze human adipose-derived mesenchymal stromal cells (hADSCs). In the first experiment, we compared the combined effects of 3% trehalose (3 T) and 5% dextran (5D) in lactated Ringer’s solution (LR) as a cryopreservation base solution containing 10% propylene glycol (PG). The cell viability of hADSCs immediately after thawing was significantly higher (p

The modulation of the tumor microenvironment by natural products may play a significant role in the response of tumor cells to chemotherapy. In this study, we evaluated the effect of extracts derived from P2Et (Caesalpinia spinosa) and Anamú-SC (Petiveria alliacea) plants, previously studied by our group, on the viability and ROS levels in the K562 cell line (Pgp− and Pgp+), endothelial cells (ECs, Eahy.926 cell line) and mesenchymal stem cells (MSC) cultured in 2D and 3D. The results show that: (a) the two botanical extracts are selective on tumor cells compared to doxorubicin (DX), (b) cytotoxicity is independent of the modulation of intracellular ROS for plant extracts, unlike DX, (c) the interaction with DX can be influenced by chemical complexity and the expression of Pgp, (d) the 3D culture shows a greater sensitivity of the tumor cells to chemotherapy, in co-treatment with the extracts. In conclusion, the effect of the extracts on the viability of leukemia cells was modified in multicellular spheroids with MSC and EC, suggesting that the in vitro evaluation of these interactions can contribute to the comprehension of the pharmacodynamics of the botanical drugs.

INTRODUCTION: The regenerative and immunomodulatory properties of multipotent mesenchymal stromal cells (MSCs) make them an intriguing asset for therapeutic applications. An off-the-shelf approach, using pre-expanded cryopreserved allogenic MSCs, bypasses many practical difficulties of cellular therapy. Reconstitution of a MSC product away from cytotoxic cryoprotectants towards a preferred administration solution might be favorable for several indications. Variations in MSC handling accompanied by a non-standardized use of reconstitution solutions complicate a general clinical standardization of MSC cellular therapies. In this study, we aimed to identify a simple and clinically compatible approach for thawing, reconstitution, and post-thaw storage of cryopreserved MSCs.METHODS: Human adipose tissue-derived MSCs were expanded in human platelet lysate (hPL) supplemented culture medium and cryopreserved using a dimethyl sulfoxide (DMSO)-based cryoprotectant. Isotonic solutions (saline, Ringer's acetate and phosphate buffered saline (PBS)) with or without 2% human serum albumin (HSA) were used as thawing, reconstitution, and storage solutions. MSCs were reconstituted to 5 × 10 6 MSCs/mL for evaluating MSC stability. Total MSC numbers and viability were determined using 7-aminoactinomycin D (7-AAD) and flow cytometry. RESULTS: For thawing cryopreserved MSCs the presence of protein was proven to be essential. Up to 50% of MSCs were lost when protein-free thawing solutions were used. Reconstitution and post-thaw storage of MSCs in culture medium and widely used PBS demonstrated poor MSC stability (>40% cell loss) and viability (90% viability with no observed cell loss for at least 4 h. Reconstitution of MSCs to low concentrations was identified as critical. Diluting MSCs to 40% cell loss) and lower viability (CONCLUSION: This study identified a clinically compatible method for MSC thawing and reconstitution that ensures high MSC yield, viability, and stability. The strength of the method lies within the simplicity of implementation which offers an accessible way to streamline MSC therapies across different laboratories and clinical trials, improving standardization in this field.

New therapeutic options for liver cirrhosis are needed. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have emerged as a promising tool for delivering therapeutic factors in regenerative medicine. Our aim is to establish a new therapeutic tool that employs EVs derived from MSCs to deliver therapeutic factors for liver fibrosis. EVs were isolated from supernatants of adipose tissue MSCs, induced-pluripotent-stem-cell-derived MSCs, and umbilical cord perivascular cells (HUCPVC-EVs) by ion exchange chromatography (IEC). To produce engineered EVs, HUCPVCs were transduced with adenoviruses that code for insulin-like growth factor 1 (AdhIGF-I-HUCPVC-EVs) or green fluorescent protein. EVs were characterized by electron microscopy, flow cytometry, ELISA, and proteomic analysis. We evaluated EVs’ antifibrotic effect in thioacetamide-induced liver fibrosis in mice and on hepatic stellate cells in vitro. We found that IEC-isolated HUCPVC-EVs have an analogous phenotype and antifibrotic activity to those isolated by ultracentrifugation. EVs derived from the three MSCs sources showed a similar phenotype and antifibrotic potential. EVs derived from AdhIGF-I-HUCPVC carried IGF-1 and showed a higher therapeutic effect in vitro and in vivo. Remarkably, proteomic analysis revealed that HUCPVC-EVs carry key proteins involved in their antifibrotic process. This scalable MSC-derived EV manufacturing strategy is a promising therapeutic tool for liver fibrosis.

Mesenchymal stem cells (MSC) can be used to facilitate reparative osteogenesis. In the case of critical-size defects, MSC can attach to allogenic bone implants (AlloI) that serve as a matrix. Objective. Analyze the morphological features of reparative osteogenesis in critical-size defects in femurs of rats (3 and 12 months old) when thedefects are filled with MSC along with AlloI. Methods. 60 white lab rats, 3 months (n=30) and 12 months (n=30) old were used. Defects (3mm in depth, 3mm in diameter) were created in thefemoral metaphysis of each rat, and filled with AlloI in the control groups and with AlloI and adipose-derived MSC in the experimental groups. Each group contained 15 rats of a particular age. 14, 28, and 90days after the surgery, histological studies were conducted. Results. The area of AlloI decreased with time. 14 days after thesurgery, in the experimental group, the area of AlloI was 1.6 times greater in 3-month-old (3mo) rats than in 12-month-old (12mo) rats. In comparison to the control, the area of AlloI was greater 14 days after surgery in 3mo rats and 28 days after surgery in 12mo rats. 14 and 28 days after the operation, the area of connective tissue was greater in rats of both experimental groups than in the control. For the 3mo rats, the same was true 90 days after the operation. Thearea of newly formed bone was 1.6 times lower in 3mo rats than in 12mo rats 14 days after the operation. 90 days after theoperation, the area was 2.3 greater in 3mo rats. For 12mo rats, thehighest area of bone tissue occurred 14 days after the surgery, and subsequently did not significantly change or differ from the control. For 3mo rats, the area of bone tissue was lower than control 14 and 28 days after the surgery, but greater than control 90 days after the surgery. Conclusions. The use of MSC along with AlloI to fill traumatic bone defects causes slower bone formation and excessive formation of connective tissue, independent of theage of the recipient., Мезенхімальні стромальні клітини (МСК) використовують для оптимізації репаративного остеогенезу. Каркасом для їхнього прикріплення можуть бути алогенні кісткові імплантати (АлоІ). Мета. Проаналізувати перебіг репаративного остеогенезу в дефектах критичного розміру стегнових кісток щурів (вік 3 і 12 міс.) за умов пластики АлоІ із МСК. Методи. Використано 60 білих лабораторних щурів віком 3міс. (n = 30) і 12 міс. (n = 30). Дефект (глибина 3мм, діаметр 3 мм) у метафізі стегнової кістки заповнили в контрольних групах АлоІ (по 15 щурів кожного віку), у дослідних— АлоІ із МСК із жирової тканини (по 15тварин кожного віку). Через 14, 28 і 90 діб після операції виконано гістологічні дослідження. Результати. Відносна площа АлоІ протягом експерименту зменшувалася. У досліді на 14-тудобу в 3-місячних щурів вона виявилася більшою в 1,6раза, ніж у 12-місячних, а порівняно зконтролем була більшою в молодших щурів на 14-ту добу, у старших— на 28-му. Відносні площі сполучної тканини за умов використання АлоІ та МСК у тварин обох вікових груп були більшими на 14- та 28-му доби, а у3-місячних щурів — іна 90-ту, ніж уразі застосування АлоІ окремо. Відносна площа новоутворених кісткових трабекул у щурів віком 3міс. на 14-тудобу була нижчою в 1,6 раза, на 90-ту— більшою у 2,3раза, ніж у12-місячних тварин. В останніх площа кісткової тканини досягла вищого показника на 14- тудобу і надалі значуще не змінювалася та не відрізнялася від контролю цього віку. У3-місячних щурів на 14- та 28-мудоби показник був нижчим приблизно в 1,5раза, ана 90- ту — більшим в 1,9раза порівняно з контролем цього віку. Висновки. Введення МСК разом із АлоІ у випадках свіжих травматичних ушкоджень кісток спричинює уповільнення кісткоутворення незалежно від віку реципієнта інадлишкове формування сполучної тканини.

The immunomodulatory properties of MSCs can be recreated using their extracellular vesicles (EVs). Yet, the true capabilities of the MSC EVs cannot be distinguished from contaminating bovine EVs and protein derived from supplemental foetal bovine serum (FBS). FBS EV depletion protocols can minimise this, but vary in terms of depletion efficiency, which can negatively impact the cell phenotype. We explore the impact of FBS EV depletion strategies, including ultracentrifugation, ultrafiltration, and serum-free, on umbilical cord MSC characteristics. Whilst a greater depletion efficiency, seen in the ultrafiltration and serum-free strategies, did not impact the MSC markers or viability, the MSCs did become more fibroblastic, had slower proliferation, and showed inferior immunomodulatory capabilities. Upon MSC EV enrichment, more particles, with a greater particle/protein ratio, were isolated upon increasing the FBS depletion efficiency, except for serum-free, which showed a decreased particle number. Whilst all conditions showed the presence of EV-associated markers (CD9, CD63, and CD81), serum-free was shown to represent a higher proportion of these markers when normalised by total protein. Thus, we caution MSC EV researchers on the use of highly efficient EV depletion protocols, showing that it can impact the MSC phenotype, including their immunomodulatory properties, and stress the importance of testing in consideration to downstream objectives.

Immune responses are highly complex and intricately regulated processes involving immune and non-immune cells in close direct and indirect contact with each other. These cells are highly sensitive to environmental signals, including factors derived from microbiota. Here, we demonstrate that the human microbiota member Lactobacillus casei (L. casei)-derived cell-free supernatant (CFS) enhances the sensitivity of mesenchymal-stromal-cell-like (MSCI) cells to viral stimuli and induces the development of dendritic cells (DCs) with anti-inflammatory and antiviral properties via pretreated MSCl cells. Our results showed that the production of INFβ and CXCL10 by MSCl cells upon viral stimulation was dependent on the presence of L. casei-derived extracellular vesicles in CFS during pretreatment. Moreover, L. casei CFS and/or poly (I:C)-conditioned MSCI cells altered the differentiation process of freshly isolated monocytes, as well as the developing DCs’ phenotype and functional activities, such as cytokine and chemokine secretion. Taken together, L. casei CFS contains factors which contribute to the pronounced antiviral response of MSCI cells, avoiding the development of inflammation via the induction of differentiation of anti-inflammatory DCs that retain their antiviral properties.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. Treatment of PDAC remains a major challenge. This study aims to evaluate, in vitro, the use of human umbilical cord mesenchymal stromal cell (UC-MSC)-derived EVs to specifically target pancreatic cancer cells. EVs were isolated from the FBS-free supernatants of the cultured UC-MSCs by ultracentrifugation and characterized by several methods. EVs were loaded with scramble or KRASG12D-targeting siRNA by electroporation. The effects of control and loaded EVs on different cell types were evaluated by assessing cell proliferation, viability, apoptosis and migration. Later, the ability of EVs to function as a drug delivery system for doxorubicin (DOXO), a chemotherapeutic drug, was also evaluated. Loaded EVs exhibited different kinetic rates of uptake by three cell lines, namely, BxPC-3 cells (pancreatic cancer cell line expressing KRASwt), LS180 cells (colorectal cell line expressing KRASG12D) and PANC-1 cells (pancreatic cell line expressing KRASG12D). A significant decrease in the relative expression of the KRASG12D gene after incubation with KRAS siRNA EVs was observed by real-time PCR. KRASG12D siRNA EVs significantly reduced the proliferation, viability and migration of the KRASG12D cell lines compared to scramble siRNA EVs. An endogenous EV production method was applied to obtain DOXO-loaded EVs. Briefly, UC-MSCs were treated with DOXO. After 24 h, UC-MSCs released DOXO-loaded EVs. DOXO-loaded EVs were rapidly taken up by PANC-1 cells and induced apoptotic cell death more efficiently than free DOXO. In conclusion, the use of UC-MSC-derived EVs as a drug delivery system for siRNAs or drugs could be a promising approach for the targeted treatment of PDAC.

Mesenchymal stromal cells (MSCs) are involved in bone tissue remodeling due to their ability to differentiate into osteoblasts and to influence osteoclasts’ activity. Multiple myeloma (MM) is associated with bone resorption. During disease progression, MSCs acquire a tumor-associated phenotype, losing their osteogenic potential. The process is associated with impaired osteoblasts/osteoclasts balance. The WNT signaling pathway plays a major role in maintaining the balance. In MM, it functions in an aberrant way. It is not known yet whether the WNT pathway is restored in patients’ bone narrow after treatment. The aim of the study was to compare the level of WNT family gene transcription in the bone marrow MSCs of healthy donors and MM patients before and after therapy. The study included healthy donors (n = 3), primary patients (n = 3) and patients with different response status to therapy (bortezomib-containing induction regimens) (n = 12). The transcription of the WNT and CTNNB1 (encoding β-catenin) genes was accessed using qPCR. The mRNA quantity of ten WNT genes, as well as CTNNB1 mRNA encoding β-catenin, a key mediator in canonical signaling, was evaluated. The observed differences between the groups of patients indicated that aberrant functioning of the WNT pathway was retained after treatment. The differences that we detected for WNT2B, WNT9B and CTNNB1 suggested their possible application as prognostic molecular markers.

Mesenchymal Stromal Cells (MSCs) derived from bone marrow are widely tested in clinical trials as a cellular therapy for potential inflammatory disorders. The mechanism of action of MSCs in mediating immune modulation is of wide interest. In the present study, we investigated the effect of human bone-marrow-derived MSCs in modulating the circulating peripheral blood dendritic cell responses through flow cytometry and multiplex secretome technology upon their coculture ex vivo. Our results demonstrated that MSCs do not significantly modulate the responses of plasmacytoid dendritic cells. However, MSCs dose-dependently promote the maturation of myeloid dendritic cells. Mechanistic analysis showed that dendritic cell licensing cues (Lipopolysaccharide and Interferon-gamma) stimulate MSCs to secret an array of dendritic cell maturation-associated secretory factors. We also identified that MSC-mediated upregulation of myeloid dendritic cell maturation is associated with the unique predictive secretome signature. Overall, the present study demonstrated the dichotomy of MSC functionality in modulating myeloid and plasmacytoid dendritic cells. This study provides clues that clinical trials need to investigate if circulating dendritic cell subsets in MSC therapy can serve as potency biomarkers.

During aging, bone marrow mesenchymal stromal cells (MSCs)—the precursors of osteoblasts—undergo cellular senescence, losing their osteogenic potential and acquiring a pro-inflammatory secretory phenotype. These dysfunctions cause bone loss and lead to osteoporosis. Prevention and intervention at an early stage of bone loss are important, and naturally active compounds could represent a valid help in addition to diet. Here, we tested the hypothesis that the combination of two pro-osteogenic factors, namely orthosilicic acid (OA) and vitamin K2 (VK2), and three other anti-inflammatory compounds, namely curcumin (CUR), polydatin (PD) and quercetin (QCT)—that mirror the nutraceutical BlastiMin Complex® (Mivell, Italy)—would be effective in promoting MSC osteogenesis, even of replicative senescent cells (sMSCs), and inhibiting their pro-inflammatory phenotype in vitro. Results showed that when used at non-cytotoxic doses, (i) the association of OA and VK2 promoted MSC differentiation into osteoblasts, even when cultured without other pro-differentiating factors; and (ii) CUR, PD and QCT exerted an anti-inflammatory effect on sMSCs, and also synergized with OA and VK2 in promoting the expression of the pivotal osteogenic marker ALP in these cells. Overall, these data suggest a potential role of using a combination of all of these natural compounds as a supplement to prevent or control the progression of age-related osteoporosis.

Let-7 miRNAs have pleiotropic cellular functions in cell proliferation, migration, and regenerative processes. Here, we investigate whether the inhibition of let-7 miRNAs with antisense oligonucleotides (ASOs) can be a transient and safe strategy enhancing the therapeutic potential of mesenchymal stromal cells (MSCs) to overcome their limitations in cell therapeutic trials. We first identified major subfamilies of let-7 miRNAs preferentially expressed in MSCs, and efficient ASO combinations against these selected subfamilies that mimic the effects of LIN28 activation. When let-7 miRNAs were inhibited with an ASO combination (anti-let7-ASOs), MSCs exhibited higher proliferation with delayed senescence during the passaging into a culture. They also exhibited increased migration and enhanced osteogenic differentiation potential. However, these changes in MSCs were not accompanied by cell-fate changes into pericytes or the additional acquisition of stemness, but instead occurred as functional changes accompanied by changes in proteomics. Interestingly, MSCs with let-7 inhibition exhibited metabolic reprogramming characterized by an enhanced glycolytic pathway, decreased reactive oxygen species, and lower transmembrane potential in mitochondria. Moreover, let-7-inhibited MSCs promoted the self-renewal of neighboring hematopoietic progenitor cells, and enhanced capillary formation in endothelial cells. These findings together show that our optimized ASO combination efficiently reprograms the MSC functional state, allowing for more efficient MSC cell therapy.

Advanced therapy medicinal products (ATMPs) offer revolutionary new prospects for the treatment of conditions of unmet medical need. Among them, Mesenchymal Stromal Cells (MSCs) have emerged as promising candidates for cell-based regenerative medical therapies. Currently, several early phase clinical trials have been completed and many are ongoing to explore MSC safety and efficacy in a wide range of nephropathologies. Kidney diseases are a current major health concern with an increasing global prevalence. The current standards of care focus on supportive treatments, emphasizing the need to develop novel and efficient therapies to delay or halt disease progression. However, before these therapies can be routinely used in patients, there are still hurdles to overcome. One of the current roadblocks relates to the lack of standardization of manufacturing protocols. Studies have shown that cell culture processing variables can have significant effects on MSC phenotype and functionality. In addition, MSCs can be isolated from various sources and there is now a growing body of literature highlighting unique and intrinsic properties according to the tissue origin and donor characteristics. Having an optimal, well-designed scale-up strategy for MSC manufacturing is critical to ensure product quality. In this thesis, we first sought to develop a protocol to expand the most clinically used MSC populations – adipose, bone marrow, and umbilical cord – in a multicentre study to compare the properties of the cells emanating from these three sources when cultured in a de-centralised manufacturing approach. By implementing a harmonised expansion workflow, we identified unique growth patterns and differentiation potential that followed comparable trends among centres. We harmonized our culture expansion conditions including the use of a single source of foetal bovine serum (FBS) in order to minimize culture condition-induced alterations in cell biology. Due to the inherent heterogeneity within the MSC-field, in terms of both culture conditions and source material, the development of strategies to select the optimal ‘source’ for a particular clinical condition would facilitate successful clinical translation. For this, the identification of features beyond basic cell characteristics are in need of further investigation. In this thesis, we focused on assessing the ability of MSCs from different tissue origin to support angiogenesis, essential during tissue regeneration. Bone marrow derived MSCs and their secretome showed significantly enhanced abilities to promote tubulogenesis and endothelial cell migration in vitro, partially due to a superior secretion of angiogenic cytokines such as vascular endothelial growth factor (VEGF). Additionally, the central metabolism is a critical driver of many cellular functions and has been shown to vary and alter the therapeutic benefit of MSCs undergoing large-expansion procedures. The comparison of metabolic characteristics and cellular fitness between MSC populations revealed a robust and stem cell-like phenotype in umbilical cord derived MSCs that conferred them higher abilities to withstand challenging microenvironments. Finally, we investigated the therapeutic efficacy of MSC-derived bioproducts, namely their secretome and extracellular vesicles, to restore metabolic disturbances in an in vitro model of renal ischaemia/reperfusion injury (IRI). Collectively, results to date emphasize the heterogeneity of MSCs in their nature and mechanism of action. The development of culture conditions appropriately designed to support cell expansion while enhancing therapeutic potential will allow for the elucidation of their unique regenerative properties, facilitating the development of tailored and efficient therapies for a given medical condition. 2025-05-08

The genetic engineering of human mesenchymal stromal cells (hMSCs) is a promising strategy to improve their therapeutic potential and clinical translation. Viral vectors based on human adenovirus type 5 (HAdV-5) are a versatile tool for this purpose. However, due to a lack of coxsackie and adenovirus receptor (CAR) expression, transduction of hMSCs with HAdV-5 vectors is naturally very inefficient, and strategies to overcome this limitation are essential for therapeutic application. In the present study, we identified and characterized several transduction enhancers which significantly improved the transduction of hMSCs with HAdV-5 vectors. Especially with the polyamines spermine and spermidine, expression levels of a reporter transgene were increased more than 1000 fold, and significantly elevated expression and secretion of a potential therapeutic protein (Tumor necrosis factor-inducible gene 6 protein (TSG-6)) was achieved. Furthermore, we found that the genetically charge-modified vector HAdV-5-HexPos3 (exchange of 13 negatively charged amino acids by four lysines in the hypervariable region 1 (HVR1) of Hexon) enabled efficient transduction of hMSCs (and several tumor cell lines) without the need for enhancing molecules. Transduction with HAdV-5-HexPos3 was even significantly higher than with the chimeric vector HAdV-5/3, which has been shown to be one of the most efficient adenovirus-based vectors for the transduction of hMSCs to date. We found that HAdV-5-HexPos3 uses a CAR-independent uptake mechanism that involves binding to heparan sulfate proteoglycans (HSPGs), while the interactions with non-cellular blood components were unaltered. In addition, we evaluated the enhanced transduction of hMSCs in two pre-clinical models of hMSC-based oncolytic virotherapy, in which hMSCs were used as carrier cells for oncolytic viruses to the tumor tissue. However, neither in two human head and neck squamous cell carcinoma (HNSCC) xenograft tumor models established in mice nor in a chorioallantoic-membrane (CAM) model migration of transduced hMSCs towards the tumor was detected. Nevertheless, in in vitro analyses, we showed that both, transduced or infected hMSCs, migrated towards several cell lines, including HNSCC cell lines. Moreover, we confirmed efficient replication of wildtype and life cycle modified HAdV-5 viruses in hMSCs, which was significantly accelerated by a knock-out mutation of the adenoviral E1B 19K protein. Strikingly, replication-competent vectors carrying the HexPos3 modification showed significantly improved replication, highlighting the potential of HAdV-5-HexPos3 for hMSC-based oncolytic approaches. In summary, transduction enhancers and the novel HAdV-5-HexPos3 mutant are valuable tools for the efficient genetic engineering of hMSCs either to overexpress therapeutic transgenes or in the context of oncolytic virotherapy. However, in vivo testing of hMSC-based oncolytic approaches requires further investment in suitable animal models.

Cartilage tissue presents low self-repair capability and lesions often undergo irreversible progression. Structures obtained by tissue engineering, such as those based in extrusion bioprinting of constructs loaded with stem cell spheroids may offer valuable alternatives for research and therapeutic purposes. Human mesenchymal stromal cell (hMSC) spheroids can be chondrogenically differentiated faster and more efficiently than single cells. This approach allows obtaining larger tissues in a rapid, controlled and reproducible way. However, it is challenging to control tissue architecture, construct stability, and cell viability during maturation. Herein, this work reports a reproducible bioprinting process followed by a successful post-bioprinting chondrogenic differentiation procedure using large quantities of hMSC spheroids encapsulated in a xanthan gum-alginate hydrogel. Multi-layered constructs are bioprinted, ionically crosslinked, and post chondrogenically differentiated for 28 days. The expression of glycosaminoglycan, collagen II and IV are observed. After 56 days in culture, the bioprinted constructs are still stable and show satisfactory cell metabolic activity with profuse extracellular matrix production. These results show a promising procedure to obtain 3D models for cartilage research and ultimately, an in vitro proof-of-concept of their potential use as stable chondral tissue implants.

Mesenchymal stromal/stem cells are multipotent adult cells that can be extracted from numerous tissues, including the lungs. Lung-resident MSCs (LR-MSCs) are localized to perivascular spaces where they act as important regulators of pulmonary homeostasis, mediating the balance between lung injury/damage and repair processes. LR-MSCs support the integrity of the lung tissue via modulation of the immune response and release of trophic factors. However, in the context of chronic lung diseases, the ability of LR-MSCs to maintain pulmonary homeostasis and facilitate repair is diminished. In this setting, LR-MSC can contribute to the pathogenesis of disease, through their altered secretory and immunomodulatory properties. In addition, they are capable of differentiating into myofibroblasts, thereby contributing to the fibrotic aspects of numerous lung diseases. For example, in idiopathic pulmonary fibrosis, a variety of factors can stimulate their differentiation into myofibroblasts including tumor necrosis factor-α (TNF-(α), transforming growth factor-β1 (TGF-β1), endoplasmic reticulum (ER) stress, Hedgehog (HH), and Wingless/integrated (Wnt) signaling. Here, we review the current literature on the characterization of LR-MSCs and describe their roles in pulmonary homeostasis/repair and in the pathogenesis of chronic lung disease.

Perinatal derivatives (PnD) are birth-associated tissues, such as placenta, umbilical cord, amniotic and chorionic membrane, and thereof-derived cells as well as secretomes. PnD play an increasing therapeutic role with beneficial effects on the treatment of various diseases. The aim of this review is to elucidate the modes of action of non-hematopoietic PnD on inflammation, angiogenesis and wound healing. We describe the source and type of PnD with a special focus on their effects on inflammation and immune response, on vascular function as well as on cutaneous and oral wound healing, which is a complex process that comprises hemostasis, inflammation, proliferation (including epithelialization, angiogenesis), and remodeling. We further evaluate the different in vitro assays currently used for assessing selected functional and therapeutic PnD properties. This review is a joint effort from the COST SPRINT Action (CA17116) with the intention to promote PnD into the clinics. It is part of a quadrinomial series on functional assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer activities, anti-inflammation, wound healing, angiogenesis, and regeneration.

Aim The variable results in clinical trials of adipose tissue-derived stem cells (ASCs) for chondral defects may be due to the different ex vivo culture conditions of the ASCs which are implanted to treat the lesions. We sought to determine the optimal in vitro chondrocyte co-culture condition that promotes infrapatellar fat pad-derived (IFPD) ASC chondrogenic gene expression in a novel co-culture combination. Methods In our study, we utilized an in vitro autologous co-culture of IFPD ASCs and articular chondrocytes derived from Kellgren–Lawrence Grade III/IV osteoarthritic human knee joints at ASC-to-chondrocyte seeding log ratios of 1:1, 10:1, and 100:1. Gene expression following in vitro co-culture was quantified by RT-qPCR with a panel comprising COL1A1, COL2A1, COL10A1, L-SOX5, SOX6, SOX9, ACAN, HSPG2, and COMP for chondrogenic gene expression. Results The chondrogenic gene expression profiles from co-cultures were greater than would be expected from an expression profile modeled from chondrocyte and ASC-only monocultures. Additionally, chondrogenic gene expression decreased with increasing ASC-to-chondrocyte seeding ratios. Conclusions These findings provide insight into the mechanisms underlying clinical ASC therapies and signifies that IFPD ASCs pre-conditioned by chondrocyte co-culture may have improved chondrogenic potential for cartilage repair. This model can help further understand IFPD ASCs in chondral and osteochondral repair and the chondrogenic pathways involved.

Background Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive deadly fibrotic lung disease with high prevalence and mortality worldwide. The therapeutic potential of mesenchymal stem cells (MSCs) in pulmonary fibrosis may be attributed to the strong paracrine, anti-inflammatory, anti-apoptosis and immunoregulatory effects. However, the mechanisms underlying the therapeutic effects of MSCs in IPF, especially in terms of alveolar type 2 (AT2) cells senescence, are not well understood. The purpose of this study was to evaluate the role of MSCs in NAD metabolism and senescence of AT2 cells in vitro and in vivo. Methods MSCs were isolated from human bone marrow. The protective effects of MSCs injection in pulmonary fibrosis were assessed via bleomycin mouse models. The senescence of AT2 cells co-cultured with MSCs was evaluated by SA-β-galactosidase assay, immunofluorescence staining and Western blotting. NAD+ level and NAMPT expression in AT2 cells affected by MSCs were determined in vitro and in vivo. FK866 and NAMPT shRNA vectors were used to determine the role of NAMPT in MSCs inhibiting AT2 cells senescence. Results We proved that MSCs attenuate bleomycin-induced pulmonary fibrosis in mice. Senescence of AT2 cells was alleviated in MSCs-treated pulmonary fibrosis mice and when co-cultured with MSCs in vitro. Mechanistic studies showed that NAD+ and NAMPT levels were rescued in AT2 cells co-cultured with MSCs and MSCs could suppress AT2 cells senescence mainly via suppressing lysosome-mediated NAMPT degradation. Conclusions MSCs attenuate AT2 cells senescence by upregulating NAMPT expression and NAD+ levels, thus exerting protective effects in pulmonary fibrosis.

Background Knee osteoarthritis (OA) is a debilitating condition affecting human body biomechanics and quality of life. Current standard care for knee OA leads to trivial improvement and entails multiple adverse effects or complications. Recently, investigational cell therapies injected intra-articularly, such as bone marrow aspirate concentrate (BMAC) and platelet-rich plasma (PRP), have shown safety and therapeutic potency providing patients with pain relief. In the current retrospective comparative study, we investigated the differences in pain and functional improvements in patients with symptomatic knee OA receiving intra-articular injections of BMAC vs PRP. Methods Pain and functionality scores were measured at baseline and at different time points post-injection over 12 months, using 3 self-administered, clinically validated questionnaires: the visual analogue scale (VAS) for assessing pain intensity, the knee injury and osteoarthritis outcome score (KOOS) for evaluating functionality and knee-related quality of life, and the Western Ontario and McMaster Universities Arthritis Index (WOMAC) for evaluating physical function. The repeated-measures general linear model with Sidak test for pairwise comparisons was used to investigate the influence of the treatment on the score evolution within groups (between baseline and each time point) and between groups (overall). Results The BMAC group (n = 26 knees) significantly improved in VAS, KOOS, and WOMAC scores between baseline and 12 months (57.4, 75.88, and 73.95% mean score improvement, respectively). In contrast, the PRP group (n = 13 knees) witnessed nonsignificant improvement in all scores. BMAC, in comparison to PRP, induced significant improvement in outcomes by 29.38% on the VAS scale, 53.89% on the KOOS scale, and 51.71% on the WOMAC scale (P P P Conclusions Intra-articular autologous BMAC injections are safe, effective in treating pain, and ameliorate functionality in patients with symptomatic knee OA to a greater extent than PRP injections. Graphical abstract Intra-articular autologous BMAC therapy is safe and provides more relief to patients with symptomatic knee osteoarthritis compared to PRP therapy.

Inflammation-associated disorders are significant causes of morbidity in horses. Equine single-donor mesenchymal stromal cells (sdMSCs) hold promise as cell-therapy candidates due to their secretory nonprogenitor functions. This has been demonstrated by mononuclear cell suppression assays (MSAs) showing that sdMSCs are blood mononuclear cell (BMC) suppressive in vitro. sdMSCs derived from umbilical cord blood are of clinical interest due to their ease of procurement, multipotency, and immunomodulatory ability. Due to the inherent donor-to-donor heterogeneity of MSCs, the development of robust and easily deployable methods of potency assessment is critical for improving MSCs' predictability in treating inflammatory diseases. This study focuses on the development of robust in vitro potency assays and the assessment of potential sdMSC therapeutic end products generated from pooled sdMSCs (pMSCs). We hypothesized that, compared to MSA using only one donor, MSA using pooled BMCs (pBMCs) is a more robust sdMSC potency assay due to reduced donor BMC heterogeneity. pBMCs were generated by pooling equine BMCs isolated from peripheral blood of five donors in equal ratios. pBMCs were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stored in liquid nitrogen until use. Similarly, pooling sdMSCs from multiple equine donors in equal ratios generated pMSCs. sdMSC cultures were assessed with pBMCs in MSA using Bromodeoxyuridine ELISA and CFSE. Proliferation assessment of BMCs from individual donors revealed varied responses to concanavalin A (ConA) stimulation. MSA using BMCs from single donors further demonstrated BMC donor variability. Utilizing this assay, we have also found that the immunosuppressive potencies of pMSCs are at least equal, if not more, than the calculated mean of individual cultures. MSA based on pBMCs provides a consistent and reproducible equine sdMSC potency assay. This knowledge could be used in production monitoring of cellular potency and as release criteria before clinical use.

Introduction: Mesenchymal stromal cells (MSCs) administration is an effective option for the treatment of diabetic foot ulcers (DFUs). However, to date, studies assessing long-term outcomes and evaluating skin parameters after cell-based therapy are lacking. We presented the clinical outcomes of 3 patients, treated for DFUs with the bone marrow MSCs 3 years earlier. Methods: Ultrasound examination was used to compare collagen density and epidermal thickness in areas of healed ulcers in comparison with non-affected skin used as a control. Ultrasound and dermatoscopy were used to exclude neoplasm formation, to assess scar contracture and wound recurrence. Results: In all patients, no ulcer recurrence was detected, which was lower than the expected 60% rate of re-ulceration in diabetic patients in a 3-year period (OD [odds ratio] = 0.095, P = 0.12). No neoplasm formation, no contracture of hypertrophic scar, and adjacent tissue were registered. Collagen ultrasound density was decreased by 57% (P = 0.053) and epidermal thickness was increased by 72% (P = 0.01) in the area of healed ulcers in all patients. Conclusion: MSCs therapy alone did not result in the complete restoration of the skin parameters within a 3-year period. MSCs may represent important adjuvant to the therapy, however, other novel approaches are required to achieve better results.

Delivery of a peptide (APP96-110), derived from amyloid precursor protein (APP), has been shown to elicit neuroprotective effects following cerebral stroke and traumatic brain injury. In this study, the effect of APP96-110 or a mutant version of this peptide (mAPP96-110) was assessed following moderate (200 kdyn, (2 N)) thoracic contusive spinal cord injury (SCI) in adult Nude rats. Animals received a single tail vein injection of APP96-110 or mAPP96-110 at 30 minutes post-SCI and were then assessed for functional improvements over the next 8 weeks. A cohort of animals also received transplants of either viable or non-viable human mesenchymal stromal cells (hMSCs) into the SC lesion site at one week post-injury to assess the effect of combining intravenous APP96-110 delivery with hMSC treatment. Rats were perfused 8 weeks post-SCI and longitudinal sections of spinal cord analyzed for a number of factors including hMSC viability, cyst size, axonal regrowth, glial reactivity and macrophage activation. Analysis of sensorimotor function revealed occasional significant differences between groups using Ladderwalk or Ratwalk tests, however there were no consistent improvements in functional outcome after any of the treatments. mAPP96-110 alone, and APP96-110 in combination with both viable and non-viable hMSCs significantly reduced cyst size compared to SCI alone. Combined treatments with donor hMSCs also significantly increased βIII tubulin+, glial fibrillary acidic protein (GFAP+) and laminin+ expression, and decreased ED1+ expression in tissues. This preliminary study demonstrates that intravenous delivery of APP96-110 peptide has selective, modest neuroprotective effects following SCI, which may be enhanced when combined with hMSC transplantation. However, the effects are less pronounced and less consistent compared to the protective morphological and cognitive impact that this same peptide has on neuronal survival and behaviour after stroke and traumatic brain injury. Thus while the efficacy of a particular therapeutic approach in one CNS injury model may provide justification for its use in other neurotrauma models, similar outcomes may not necessarily occur and more targeted approaches suited to location and severity are required. All animal experiments were approved by The University of Western Australia Animal Ethics Committee (RA3/100/1460) on April 12, 2016.

Each person is inevitably exposed to low doses of ionizing radiation (LDIR) throughout their life. The research results of LDIR effects are ambiguous and an accurate assessment of the risks associated with the influence of LDIR is an important task. Mesenchymal stromal cells (MSCs) are the regenerative reserve of an adult organism; because of this, they are a promising model for studying the effects of LDIR. The qualitative and quantitative changes in their characteristics can also be considered promising criteria for assessing the risks of LDIR exposure. The MSCs from human connective gingiva tissue (hG-MSCs) were irradiated at doses of 50, 100, 250, and 1000 mGy by the X-ray unit RUST-M1 (Russia). The cells were cultured continuously for 64 days after irradiation. During the study, we evaluated the secretory profile of hG-MSCs (IL-10, IDO, IL-6, IL-8, VEGF-A) using an ELISA test, the immunophenotype (CD45, CD34, CD90, CD105, CD73, HLA-DR, CD44) using flow cytometry, and the proliferative activity using the xCelligence RTCA cell analyzer at the chosen time points. The results of study have indicated the development of stimulating effects in the early stages of cultivation after irradiation using low doses of X-ray radiation. On the contrary, the effects of the low doses were comparable with the effects of medium doses of X-ray radiation in the long-term periods of cultivation after irradiation and have indicated the inhibition of the functional activity of MSCs.

Despite osteoarthritis (OA) representing a large burden for health and social care systems, affecting approximately 10 million people in the UK alone, there remains no effective intervention capable of regenerating the damaged joint cartilage in OA and current clinical management for end-stage disease remains joint replacement surgery. There is therefore a need for a change in healthcare strategies to manage early disease, a shift that is likely to include cell-based regenerative therapies. Regenerative medicine and cell therapies are poised to have a tremendous impact on the future of medicine by delivering more effective and personalised treatment than are currently available today. Mesenchymal stromal cells (MSCs) are adult-derived, multipotent cells which have immunomodulatory effects, and can differentiate into a variety of cell types, including cartilage progenitor cells, making MSCs a candidate for musculoskeletal cell therapy. However, there has been several challenges to translating basic science research into new therapies and two pivotal questions remain unanswered: • Where does the MSC therapy go after administration? • What is its precise mechanism of action? To address these questions, the effects of an intra-articular injection of human bone marrow derived MSCs into a mouse knee osteochondral injury model were investigated in young C57Bl/6 mice, aged C57Bl/6 mice and GNL3 heterozygote mice. MSC treatment was associated with improved osteochondral tissue repair in young C57Bl/6 mice but this was more limited in aged C57BL/6 mice and GNL3 heterozygote mice. Cage activity monitoring was used to assess mouse recovery following surgery, and the administration of human MSCs was associated with significantly improved recovery following injury when compared to untreated controls. To better understand the mechanisms of action of an MSC therapy, a novel gene reporter system which utilises the organic anion transporting protein, Oatp1a1, was used for cell tracking. Using a lentiviral vector system, transfected MSCs expressing this cell surface protein can transport several imageable small molecules across the cell membrane, including the MRI contrast agent, gadolinium-ethoxybenzyl-DTPA (Gd-EOB-DTPA, PrimovistTM) which is licensed for clinical use. Cells were successfully imaged in vitro and in vivo as a proof-of-concept for this approach in the musculoskeletal system. Whilst imaging provided circumstantial evidence of MSC therapy fate, single cell RNAseq of retrieved cells (following injection into the mouse after osteochondral injury) was performed to elucidate what transcriptomic changes were important to driving tissue repair over time. Here, the data demonstrated that the exogenous human MSC therapy can be retrieved from both the repair tissue (in the epiphysis) and synovial tissue of the damaged knee joint at both 1 and 4 weeks after intra-articular injection. The retrieved human MSC therapy from the mouse joint tissue showed spatial and temporal transcriptional heterogeneity which was pronounced between the two tissue sources. Supported by mass cytometry data, the paracrine role of MSC treatment was further emphasises, as MSC therapy was associated with the induction of regulatory T cells (Tregs) in the mouse knee joint tissues. As Tregs are involved in skeletal homeostasis, more work needs to be performed to investigate the crosstalk that exists between MSCs and Tregs. Overall, the presented work shows that exogenous human MSC therapy in the mouse may improve both histological and clinical outcomes following osteochondral injury. Following intra-articular injection of human MSCs, the transcriptomes of the retrieved human cells were studied for the first time and their heterogeneity described. Subpopulations with different functional roles may be implicated in the different phases of osteochondral repair. As tissue digestion and isolation protocols are refined, it is likely that rarer populations may be retrieved from the mouse osteochondral model, but the data presented here offers insights into the interaction between the MSC therapy and the host cells, opening new avenues for the role which MSCs can play as a cell therapy in osteochondral repair., Addenbrooke's Charitable Trust/Cambridge Biomedical Research Centre; Wellcome Trust

The muscle stem cell (MuSC) population is recognized as functionally heterogeneous. Cranial muscle stem cells, which originate from head mesoderm, can have greater proliferative capacity in culture and higher regenerative potential in transplantation assays when compared to those in the limb. The existence of such functional differences in phenotypic outputs remain unresolved as a comprehensive understanding of the underlying mechanisms is lacking. We addressed this issue using a combination of clonal analysis, live imaging, and scRNA-seq, identifying critical biological features that distinguish extraocular (EOM) and limb (Tibialis anterior, TA) MuSC populations. Time-lapse studies using aMyogenintdTomatoreporter showed that the increased proliferation capacity of EOM MuSCs is accompanied by a differentiation delayin vitro. Unexpectedly, in vitro activated EOM MuSCs expressed a large array of distinct extracellular matrix (ECM) components, growth factors, and signaling molecules that are typically associated with mesenchymal non-muscle cells. These unique features are regulated by a specific set of transcription factors that constitute a coregulating module. This transcription factor network, which includes Foxc1 as one of the major players, appears to be hardwired to EOM identity as it is present in quiescent adult MuSCs, in the activated counterparts during growth and retained upon passages in vitro. These findings provide insights into how high-performing MuSCs regulate myogenic commitment by active remodeling of their local environment.

Acute respiratory distress syndrome (ARDS) is a devastating critical illness that holds the highest cost of any acute care condition in Canada. In ARDS, a localized inflammatory response caused by lung infection or injury leads to vascular damage and migration of inflammatory cells into the airways resulting in impaired gas exchange and respiratory failure. Despite decades of research, there are no curative therapies for this daunting acute lung condition. Animal studies have demonstrated a potential for mesenchymal stromal cells (MSCs) to reduce inflammation, promote tissue regeneration and improve survival in acute lung injury (ALI, the preclinical correlate of ARDS). In particular, a growing body of evidence suggests that MSCs elicit their protective effects in large part by secretion of small membrane-bound particles known as "extracellular vesicles". These vesicles carry biologically active cargo that modulate critical cell processes including programmed cell death, proliferation and inflammation. Hence, MSC-derived extracellular vesicles (MSC-EVs) may have advantages as a safer off-the-shelf, cell-free immune modulatory therapy. However, the optimal approach for isolating and administering MSC-EVs have yet to be established within this new field of regenerative medicine. The aims of this project were to: (1) evaluate the current preclinical evidence on methodology and efficacy of MSC-EVs, (2) determine the effectiveness of MSC-EVs for ALI and other pulmonary diseases in animal models, and (3) explore the kinetics and biodistribution of MSC-EVs during healthy and disease state as a prerequisite to designing optimal therapeutic strategies. By combining both clinical meta-research methodology and basic science techniques, I hoped to create a comprehensive evidence map of mechanistic insights and the therapeutic benefits of MSC-EVs. Importantly, ARDS is recognized as the main cause of mortality for patients with COVID-19. In response to the SARS-CoV2 pandemic and restrictions placed on routine, non-COVID related lab work, an additional objective for my PhD focused on developing a novel, more accessible mouse model of COVD-19 associated ARDS. A SARS-CoV2 "pseudovirus" was created to enable its use in a less stringent Containment Level 2 lab setting. Dose-escalation experiments in mice helped determine the optimal dose that produces clinically-relevant COVID-19 ARDS pathology. Moreover, the specificity of our pseudovirus to human ACE2 receptors was tested using preclinical models. If successful, this model will allow researchers worldwide to study key mechanisms in COVID-19 pathophysiology and investigate novel vaccines or therapies to combat this global pandemic.

Mesenchymal stromal cells (MSC) are part of the bone marrow architecture and contribute to the homeostasis of hematopoietic stem cells. Moreover, they are known to regulate immune effector cells. These properties of MSC are pivotal under physiologic conditions, and they may aberrantly also protect malignant cells. MSCs are also found in the leukemic stem cell niche of the bone marrow and as part of the tumor microenvironment. Here, they protect malignant cells from chemotherapeutic drugs and from immune effector cells in immunotherapeutic approaches. Modulation of these mechanisms may improve the efficacy of therapeutic regimens. We investigated the effect of the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA, Vorinostat™) on the immunomodulatory effect and cytokine profile of MSC derived from bone marrow and pediatric tumors. The immune phenotype of MSC was not markedly affected. SAHA-treated MSC showed reduced immunomodulatory effects on T cell proliferation and NK cell cytotoxicity. This effect was accompanied by an altered cytokine profile of MSC. While untreated MSC inhibited the production of certain pro-inflammatory cytokines, SAHA treatment led to a partial increase in IFNγ and TNFα secretion. These alterations of the immunosuppressive milieu might be beneficial for immunotherapeutic approaches.

Despite a long history of investigation and sustained efforts in clinical testing with large numbers of clinical trials being initiated, the number of market authorisations for mesenchymal stromal cell (MSC) therapies remains limited, with none approved by the United States Food and Drug Administration (FDA). There are a multitude of barriers impeding the clinical progression of MSC therapies, to the forefront of these is the lack of standardised and consistent manufacturing protocols as well as the lack of biologically meaningful cell characterisation tools and standard release assays. A look at clinical trial registries demonstrates the diversity of MSC expansion protocols with variabilities in cell source, isolation method and expansion medium, amongst other culture variables, making it extraordinarily difficult to compare study outcomes. Current identification and characterisation standards are insufficient; they are not specific to MSCs and do not indicate cell function or therapeutic action. This work analysed the influence of five widely used culture media formulations on the isolation efficiency, growth parameters, trilineage differentiation potential and immunomodulatory potential of human bone marrow derived MSCs (BM-MSCs) and revealed significant differences in these parameters in response to the composition of the culture medium. However, analysis of surface immunophenotype showed that despite their biological differences, all cell preparations uniformly and strongly expressed the standard positive markers proposed for BM-MSCs; CD73, CD90 and CD105, a clear demonstration of the futility of the current standard. Further characterisation of the surface immunophenotype using a high-content screening panel of 243 antibodies revealed that the culture medium also influenced the surface proteome, with one third of markers assessed exhibiting variable expression profiles in response to culture medium and donor. Calculation of correlation coefficients between the aforementioned functional in vitro data and this phenotypic analysis identified a number of putative BM-MSC surface markers related to biological function for further investigation. Principal component analysis confirmed that BM-MSCs isolated and expanded in a proprietary serum-free medium previously developed in this laboratory, Purstem 2 (PS2), displayed the most consistent cell phenotypes with little variability between donors compared to platelet lysate (PL) and fetal bovine serum (FBS)-containing media. Given the safety and reproducibility issues surrounding the use of undefined media supplements such as PL and FBS, there is a need to move towards serum-free and chemically-defined culture media. PS2 medium supports the isolation and expansion of BM-MSCs from whole bone marrow but does not support isolation from the mononuclear cell (MNC) fraction of marrow only or of adipose derived stromal cells (ASCs). To address these shortcomings and develop a new iteration of the PS2 medium, growth factor and extracellular matrix molecule receptors expressed by BM-MSCs were identified and from these, candidate growth factors platelet derived growth factor (PDGF)-AA, PDGF-BB and epidermal growth factor (EGF) and an attachment factor fibronectin were selected for analysis. The three growth factors were added alone and in combinations to the existing PS2 and tested for their ability to isolate ASCs from the stromal vascular fraction (SVF) of adipose tissue with fibronectin coating of the tissue culture plates. Isolation and propagation of ASCs was successful in all media formulations containing PDGF-BB. Assessment of cell morphology, growth kinetics, trilineage differentiation propensity, immunomodulatory potential and surface immunophenotype demonstrated equivalent or greater performance in the serum/xeno-free media compared to 5% PL controls. However, these media did not support BM-MSC isolation from the MNC fraction of bone marrow only, indicating distinct biological differences between MSCs derived from different tissues. This highlights a deficit in our understanding of the biology of MSCs derived from different tissue sources and the need for bespoke culture media. 2025-02-28

Functionalizing biomaterials with conditioned media (CM) from mesenchymal stromal cells (MSC) is a promising strategy for enhancing the outcomes of guided bone regeneration (GBR). This study aimed to evaluate the bone regenerative potential of collagen membranes (MEM) functionalized with CM from human bone marrow MSC (MEM-CM) in critical size rat calvarial defects. MEM-CM prepared via soaking (CM-SOAK) or soaking followed by lyophilization (CM-LYO) were applied to critical size rat calvarial defects. Control treatments included native MEM, MEM with rat MSC (CEL) and no treatment. New bone formation was analyzed via micro-CT (2 and 4 weeks) and histology (4 weeks). Greater radiographic new bone formation occurred at 2 weeks in the CM-LYO group vs. all other groups. After 4 weeks, only the CM-LYO group was superior to the untreated control group, whereas the CM-SOAK, CEL and native MEM groups were similar. Histologically, the regenerated tissues showed a combination of regular new bone and hybrid new bone, which formed within the membrane compartment and was characterized by the incorporation of mineralized MEM fibers. Areas of new bone formation and MEM mineralization were greatest in the CM-LYO group. Proteomic analysis of lyophilized CM revealed the enrichment of several proteins and biological processes related to bone formation. In summary, lyophilized MEM-CM enhanced new bone formation in rat calvarial defects, thus representing a novel ‘off-the-shelf’ strategy for GBR.

Methyl-CpG binding protein 2 (MeCP2) is a ubiquitous transcriptional regulator. The study of this protein has been mainly focused on the central nervous system because alterations of its expression are associated with neurological disorders such as Rett syndrome. However, young patients with Rett syndrome also suffer from osteoporosis, suggesting a role of MeCP2 in the differentiation of human bone marrow mesenchymal stromal cells (hBMSCs), the precursors of osteoblasts and adipocytes. Here, we report an in vitro downregulation of MeCP2 in hBMSCs undergoing adipogenic differentiation (AD) and in adipocytes of human and rat bone marrow tissue samples. This modulation does not depend on MeCP2 DNA methylation nor on mRNA levels but on differentially expressed miRNAs during AD. MiRNA profiling revealed that miR-422a and miR-483-5p are upregulated in hBMSC-derived adipocytes compared to their precursors. MiR-483-5p, but not miR-422a, is also up-regulated in hBMSC-derived osteoblasts, suggesting a specific role of the latter in the adipogenic process. Experimental modulation of intracellular levels of miR-422a and miR-483-5p affected MeCP2 expression through direct interaction with its 3′ UTR elements, and the adipogenic process. Accordingly, the knockdown of MeCP2 in hBMSCs through MeCP2-targeting shRNA lentiviral vectors increased the levels of adipogenesis-related genes. Finally, since adipocytes released a higher amount of miR-422a in culture medium compared to hBMSCs we analyzed the levels of circulating miR-422a in patients with osteoporosis—a condition characterized by increased marrow adiposity—demonstrating that its levels are negatively correlated with T- and Z-scores. Overall, our findings suggest that miR-422a has a role in hBMSC adipogenesis by downregulating MeCP2 and its circulating levels are associated with bone mass loss in primary osteoporosis.

The stiffness of extracellular matrices (ECMs) is critical for cellular functions. Therefore, modulating the stiffness of ECMs in vitro is necessary to investigate the role of stiffness in cellular phenomena. Collagen gels are widely used for cell culture matrices in vitro. However, modulation of the stiffness in collagen gels for cell culture is challenging owing to the limited knowledge of the method to increase the stiffness while maintaining low cytotoxicity. Here, we established a novel method to modulate collagen gel stiffness from 0.0292 to 12.5 kPa with low cytotoxicity. We prepared collagens with genipin, a low-cytotoxic crosslinker of amines, at different concentrations and successfully modulated the stiffness of the gels. In addition, on 10 mM genipin-mixed collagen gels (approximately 12.5 kPa), H1299 human lung cancer cells showed spreading morphology and nuclear localization of yes-associated protein (YAP), typical phenomena of cells on stiff ECMs. Mouse mesenchymal stromal cells on 10 mM genipin-mixed collagen gels differentiated to vascular smooth muscle cells. On the other hand, the cells on 0 mM genipin-mixed collagen gels (approximately 0.0292 kPa) differentiated to visceral smooth muscle cells. Our new method provides a novel way to prepare stiffness-modulated collagen gels with low cytotoxicity in cell culture.

Osteoarthritis (OA) is described as a chronic degenerative disease characterized by the loss of articular cartilage. Senescence is a natural cellular response to stressors. Beneficial in certain conditions, the accumulation of senescent cells has been implicated in the pathophysiology of many diseases associated with aging. Recently, it has been demonstrated that mesenchymal stem/stromal cells isolated from OA patients contain many senescent cells that inhibit cartilage regeneration. However, the link between cellular senescence in MSCs and OA progression is still debated. In this study, we aim to characterize and compare synovial fluid MSCs (sf-MSCs), isolated from OA joints, with healthy sf-MSCs, investigating the senescence hallmarks and how this state could affect cartilage repair. Sf-MSCs were isolated from tibiotarsal joints of healthy and diseased horses with an established diagnosis of OA with an age ranging from 8 to 14 years. Cells were cultured in vitro and characterized for cell proliferation assay, cell cycle analysis, ROS detection assay, ultrastructure analysis, and the expression of senescent markers. To evaluate the influence of senescence on chondrogenic differentiation, OA sf-MSCs were stimulated in vitro for up to 21 days with chondrogenic factors, and the expression of chondrogenic markers was compared with healthy sf-MSCs. Our findings demonstrated the presence of senescent sf-MSCs in OA joints with impaired chondrogenic differentiation abilities, which could have a potential influence on OA progression.

IntroductionPoor graft function (PGF) is a rare but serious complication of allogeneic hematopoietic cell transplantation (alloHCT). Due to their hematopoietic supporting properties and immune regulatory effects, multipotent mesenchymal stromal cells (MSC) could be considered a good candidate to help to restore bone marrow (BM) niches homeostasis and facilitate hematopoiesis after alloHCT.MethodsWe prospectively assessed the efficacy and safety of ex-vivo expanded BM-derived MSC from third-party donor in a series of 30 patients with prolonged severe cytopenia and PGF after alloHCT. This multicenter trial was registered at www.clinicaltrials.gov (#NTC00603330).ResultsWithin 90 days post-MSC infusion, 53% (95% CI, 35 – 71%) of patients improved at least one cytopenia (overall response, OR) and 37% (95% CI, 19 - 54%) achieved a complete hematological response (CR: absolute neutrophil count, ANC >0.5 x 109/L, Hb > 80g/L and platelet count > 20 x 109/L with transfusion independence). Corresponding response rates increased to 67% (95% CI, 50 - 84%) OR and 53% (95% CI, 35 - 71%) CR within 180 days after MSC infusion. A significant decrease in red blood cells and platelets transfusion requirement was observed after MSC (median of 30-days transfusion requirement of 0.5 and 0 from d90-120 post-MSC versus 5 and 6.5 before MSC, respectively, p ≤0.001). An increase in ANC was also noted by day +90 and +180, with 3/5 patients with severe neutropenia having recovered an ANC > 1 x 109/L within the 90-120 days after MSC infusion. Overall survival at 1 year post-MSC was 70% (95% CI, 55.4 – 88.5), with all but one of the patients who achieved CR being alive. A single infusion of third-party MSC appeared to be safe, with the exception of one deep vein thrombotic event possibly related to the intervention.DiscussionIn conclusion, a single i.v. infusion of BM-derived MSC from third party donor seemed to improve hematological function after alloHCT, although spontaneous amelioration cannot be excluded. Comparative studies are warranted to confirm these encouraging results.

Chronic myeloid leukemia (CML), BCR-ABL1-positive, is classified as a myeloproliferative characterized by Philadelphia chromosome/translocation t(9;22) and proliferating granulocytes. Despite the clinical success of tyrosine kinase inhibitors (TKi) agents in the treatment of CML, most patients have minimal residual disease contained in the bone marrow microenvironment, within which stromal cells assume a pro-inflammatory phenotype that determines their transformation in cancer-associated fibroblasts (CAF) which, in turn can play a fundamental role in resistance to therapy. Insulin-like Growth Factor Binding Protein-6 (IGFBP-6) is expressed during tumor development, and is involved in immune-escape and inflammation as well, providing a potential additional target for CML therapy. Here, we aimed at investigating the role of IGFBP-6/SHH/TLR4 axis in TKi response. We used a CML cell line, LAMA84-s, and healthy bone marrow stromal cells, HS-5, in mono- or co-culture. The two cell lines were treated with Dasatinib and/or IGFBP-6, and the expression of inflammatory markers was tested by qRT-PCR; furthermore, expression of IGFBP-6, TLR4 and Gli1 were evaluated by Western blot analysis and immumocytochemistry. The results showed that both co-culture and Dasatinib exposure induce inflammation in stromal and cancer cells so that they modulate the expression of TLR4, and these effects were more marked following IGFBP-6 pre-treatment suggesting that this molecule may confer resistance through the inflammatory processes. This phenomenon was coupled with sonic hedgehog (SHH) signaling. Indeed, our data also demonstrate that HS-5 treatment with PMO (an inducer of SHH) induces significant modulation of TLR4 and overexpression of IGFPB-6 suggesting that the two pathways are interconnected with each other and with the TLR-4 pathway. Finally, we demonstrated that pretreatment with IGFBP-6 and/or PMO restored LAMA-84 cell viability after treatment with Dasatinib, suggesting that both IGFBP-6 and SHH are involved in the resistance mechanisms induced by the modulation of TLR-4, thus indicating that the two pathways may be considered as potential therapeutic targets.

Mesenchymal stromal cells (MSCs) have been proposed as a new therapeutic strategy to treat congenital and acquired respiratory system diseases. We describe a case report of an 18-month-old male patient with progressive chronic respiratory failure, associated with mutations of the surfactant protein C gene (SFTPC) due to c.289G > T variant p.Gly97Ser (rs927644577) and c.176A > G variant (p.His59Arg), submitted to repeated intravenous infusions of allogeneic bone marrow (BM) MSCs. The clinical condition of the patient was monitored. Immunologic studies before and during MSC treatment were performed. No adverse events related to the MSC infusions were recorded. Throughout the MSC treatment period, the patient showed a growth recovery. Starting the second infusion, the patient experienced an improvement in his respiratory condition, with progressive adaptation to mechanical ventilation. After the third infusion, five hours/die of spontaneous breathing was shown, and after infusion IV, spontaneous ventilation for 24/24 h was recorded. A gradual decrease of lymphocytes and cell subpopulations was observed. No variations in the in vitro T cell response to PHA were determined by MSC treatment as well as for the in vitro B cell response. A decrease in IFN-γ, TNF-α, and IL-10 levels was also detected. Even though we cannot exclude an improvement of pulmonary function due to the physiological maturation, the well-known action of MSCs in the repair of lung tissue, together with the sequence of events observed in our patient, may support the therapeutic role of MSCs in this clinical condition. However, further investigations are necessary to confirm the result and long-term follow-up will be mandatory to confirm the benefits on the pulmonary condition.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases (prevalence 0.5-1.0%), which can lead to pain and a considerable loss of life quality in affected patients. Many details of underlying causes and mechanisms are still elusive. The persistent autoimmune-mediated inflammation of the joint is one of the key features of this systemic, chronic-inflammatory disease accounting for progressive cartilage destruction. Despite major progress in the treatment of RA, a strong unmet medical need remains. Therefore, a better understanding of the underlying pathomechanisms driving RA progression is required to develop new therapeutic strategies to effectively treat patients at every stage of disease progression. Although various RA models already exist, they either employ phylogenetically distant species or rely on human cells cultured in an oversimplified environment. To date, none of these models allows sufficient or complete extrapolation to the human patient. Therefore, the use of human-based in vitro 3D tissue equivalents of an artificial joint is a promising alternative approach to investigate pathomechanisms and test new therapeutic approaches. Hence, the aim of this thesis was to develop and characterize human in vitro 3D tissue equivalents of the joint, namely (i) cancellous bone, (ii) articular cartilage, and (iii) synovial membrane using bone marrow-derived mesenchymal stromal cells (MSCs). Here, MSCs provide the possibility of producing a complete joint model from single donor material thus offering the opportunity of a personalized testing platform. The results described in this thesis show the potential of mimicking key features of arthritis in three different tissues of a joint. Firstly, to simulate the bone component, β-tricalcium phosphate (TCP) – mimicking the mineral bony part – was populated with MSCs. Cell seeding was optimized using cell sheet technology. In contrast, the cartilage component was produced by cellular self-assembly without any supporting materials. Both models exhibit phenotypic features of native tissue, including expression of bone- or cartilage-related markers, mineralization of bone that was absent in cartilage, and development of distinct zones in the glycosaminoglycan-rich cartilage model. Co-cultivation of both tissue models generated the osteochondral unit characterized by inter-tissue connectivity, cell colonization, and initial calcification implying a functional transitional bridging area. Finally, the synovial membrane model was generated based on a xeno-free synthetic hydrogel populated with MSCs indistinguishable from synovial fibroblasts with regard to classical markers. Similar to the human synovial membrane, a confluent layer of up to four cell layers was detectable within the hydrogel allowing immune cell migration. To simulate inflammation, the individual tissue components were treated with cytokines relevant for RA, such as interleukin-6, tumor necrosis factor-α, and macrophage migration inhibitory factor. This resulted in cytokine-driven cell- and matrix-related changes in accordance with those observed during RA, while treatment using biologics prevented the induction of arthritis in the osteochondral model. These results confirm the pathophysiological mutability, architecture, integrity, and viability of the distinct in vitro 3D joint components. Prospectively, the complete in vitro 3D joint model will serve as a preclinical test platform in basic and applied biomedical research to (i) study pathophysiological processes of musculoskeletal diseases, (ii) identify new potential targets, (iii) test novel therapeutic strategies, including biologic therapies, and (iv) reduce the number of animal experiments., Rheumatoide Arthritis (RA) ist eine der häufigsten Autoimmunerkrankungen (Prävalenz 0,5-1,0 %), die bei den betroffenen Patienten zu Schmerzen und einem erheblichen Verlust an Lebensqualität führen kann. Zugrundeliegende Ursachen und Mechanismen sind nach wie vor ungeklärt. Die anhaltende autoimmunvermittelte Entzündung des Gelenks ist eines der Hauptmerkmale dieser systemisch, chronisch-entzündlichen Gelenkerkrankung. Trotz erheblicher Fortschritte bei der Behandlung der Betroffenen besteht nach wie vor eine große medizinische Versorgungslücke. Ein besseres Verständnis der zugrundeliegenden Pathomechanismen ist daher die Grundvoraussetzung für die Entwicklung neuer Therapieansätze. Bisher wurden zahlreiche RA-Modelle entwickelt, jedoch basieren diese entweder auf phylogenetisch unterschiedlichen Spezies oder auf stark vereinfachten humanen in vitro Kulturen. Bis heute lassen sich die Ergebnisse aus diesen Modellen nicht ausreichend oder vollständig auf den Patienten extrapolieren. Die Verwendung von humanen in vitro 3D Gewebeäquivalenten des Gelenks ist daher ein vielversprechender präklinischer Ansatz zur Untersuchung von Pathomechanismen und zur Prüfung neuer Therapien. Ziel dieser Arbeit war es, basierend auf humanen mesenchymalen Stromazellen (MSCs), in vitro 3D Gewebeäquivalente des Gelenks zu entwickeln. Diese Gewebeäquivalente umfassen (i) spongiösen Knochen, (ii) Gelenkknorpel und (iii) Synovialmembran. MSCs bieten hierbei die Möglichkeit, ein Gelenkmodell ausgehend vom Material eines einzelnen Spenders zu entwickeln. Das 3D Knochenmodell basiert auf dem Trägermaterial β-Trikalziumphosphat (TCP), das den mineralischen Knochenanteil des Gelenkes simuliert. Die Aussaat von Zellen auf das Trägermaterial TCP wurde mittels ‚sheet technology‘ optimiert. Im Gegensatz dazu wurde das 3D Knorpelmodell durch zelluläre Selbstorganisation, frei von zusätzlichen Trägermaterialen erzeugt. Beide Modelle weisen dem nativen Gewebe ähnelnde phänotypische Merkmale auf, wie z.B. die Expression von Knochen- oder Knorpel-spezifischen Markern, die Mineralisierung des Knochens, die im Knorpel fehlte, und die Entwicklung charakteristischer Zonen des Glykosaminoglykan-reichen Knorpelmodells. Zudem generierte die Co-Kultivierung beider Modelle eine osteochondrale Einheit, die durch Kolonisierung, Konnektivität und initiale Kalzifizierung gekennzeichnet war. Diese Eigenschaften deuten auf einen funktionellen Übergangsbereich beider Gewebeäquivalente hin. Schließlich wurde das xeno-freie Synovialmembranmodell basierend auf einem synthetischen Hydrogel entwickelt. Da sowohl MSCs als auch synovialen Fibroblasten anhand klassischer Marker nicht unterscheidbar sind, wurden MSCs in das Hydrogel eingebracht. Das Synovialmembranmodell wies eine konfluente Schicht von bis zu vier Zellschichten auf, was der physiologischen Struktur der humanen Synovialmembran entspricht. Das Hydrogel ermöglicht zudem die Migration von Immunzellen; die Voraussetzung für die Simulation des inflammatorischen Zustandes. Final wurden RA-relevante Zytokine wie Interleukin-6, Tumornekrosefaktor-α und Makrophagen-Migrationshemmungsfaktor appliziert, um Entzündungsprozesse in den Gewebeäquivalenten zu simulieren. Die Zytokin-vermittelte Stimulation resultierte in Zell- und Matrix-Veränderungen, wie sie auch bei RA beobachtet werden können. Die therapeutische Intervention hingegen führte zur Reduktion der Ausprägung der Arthritis im osteochondralen Modell. Diese Ergebnisse bestätigen die pathophysiologische Variabilität, Architektur, Integrität und Lebensfähigkeit der verschiedenen in vitro 3D Gelenkkomponenten. Perspektivisch soll das kombinierte in vitro 3D Gelenkmodell als präklinische Testplattform in der Grundlagenforschung und angewandten biomedizinischen Forschung dienen, um schlussendlich (i) pathophysiologische Prozesse muskuloskelettaler Erkrankungen studieren zu können, (ii) neue potenzielle Zielmoleküle zu identifizieren, (iii) neue therapeutische Strategien einschließlich Therapien mit Biologika zu testen und (iv) Tierversuche zu reduzieren.

Применение мезенхимальных стромальных клеток (МСК) для научных исследований и клеточной терапии требует их выращивания в достаточных количествах in vitro. В качестве источника предшественников МСК в настоящее время наиболее часто используется костный мозг. Выделение фракции мононуклеаров из костного мозга возможно несколькими способами. Целью данного исследования является сравнение градиентного и седиментационного методов выделения мононуклеаров из костного мозга, предназначенных для дальнейшего получения МСК. В настоящей работе проведено сравнение результатов выделения мононуклеаров у 25 здоровых лиц и 45 больных множественной миеломой с применением градиента плотности Lympholite-H и 0,2%-ного раствора метилцеллюлозы. Последующий посев в питательную среду позволяет получить первичную культуру МСК. Проведены расчет эффективности выделения мононуклеаров, а также оценка функциональной активности выделенных клеток путем определения количества колониеобразующих единиц фибробластов. Морфологические признаки полученной культуры и иммунофенотипическая характеристика клеток соответствовали минимальным критериям отнесения их к МСК, которые установлены Международным обществом клеточной терапии. Эффективность фракционирования костного мозга с применением обоих методов сопоставима у здоровых лиц, тем не менее колониеобразующая способность клеток выше при использовании градиента плотности. У больных множественной миеломой предпочтителен метод выделения мононуклеаров с применением метилцеллюлозы., The use of mesenchymal stromal cells (MSCs) for research and cell therapy requires their cultivation in sufficient quantities in vitro. Bone marrow is currently the most common source of MSC precursors. There are several ways to isolate mononuclear cell fraction from the bone marrow. This study is aimed to compare two MSC separation techniques, density gradient separation and sedimentation methods for the isolation of mononuclear cells from the bone marrow. In this study we compare results of isolation of mononuclear cells in 25 healthy individuals and 45 patients with multiple myeloma using Lympholite-H density gradient and 0.2% methylcellulose solution. Subsequent seeding of the obtained cells allows to get a sufficient number of MSCs. We have evaluated the efficiency of mononuclear cell isolation and the functional activity of isolated cells by determining the number of colony-forming units of fibroblasts. The morphological features of the obtained culture and the immunophenotypic characteristics of the cells meet the minimum criteria defined by International Society for Cellular Therapy for classifying them as MSCs. Both methods of bone marrow fractionation show efficiency in healthy individuals, but colony-forming ability is higher with density gradient centrifugation. The isolation of mononuclear cells using methyl-cellulose is preferable in multiple myeloma patients.

Healing large bone defects remains challenging in orthopedic surgery and is often associated with poor outcomes and complications. A major issue with bioengineered constructs is achieving a continuous interface between host bone and graft to enhance biological processes and mechanical stability. In this study, we have developed a new bioengineering strategy to produce oriented biocompatible 3D PLGA/aCaP nanocomposites with enhanced osseointegration. Decellularized scaffolds -containing only extracellular matrix- or scaffolds seeded with adipose-derived mesenchymal stromal cells were tested in a mouse model for critical size bone defects. In parallel to micro-CT analysis, SAXS tensor tomography and 2D scanning SAXS were employed to determine the 3D arrangement and nanostructure within the critical-sized bone. Both newly developed scaffold types, seeded with cells or decellularized, showed high osseointegration, higher bone quality, increased alignment of collagen fibers and optimal alignment and size of hydroxyapatite minerals., Biomaterials, 294

Tissue engineering bone via endochondral ossification requires the generation of a cartilage template which undergoes vascularisation and remodelling. While this is a promising route for bone repair, achieving effective cartilage vascularisation remains a challenge. Here, we investigated how mineralisation of tissue-engineered cartilage affects its pro-angiogenic potential. To generate in vitro mineralised cartilage, human mesenchymal stromal cell (hMSC)-derived chondrogenic pellets were treated with β-glycerophosphate (BGP). After optimising this approach, we characterised the changes in matrix components and pro-angiogenic factors by gene expression analysis, histology and ELISA. Human umbilical vein endothelial cells (HUVECs) were exposed to pellet-derived conditioned media, and migration, proliferation and tube formation were assessed. We established a reliable strategy to induce in vitro cartilage mineralisation, whereby hMSC pellets are chondrogenically primed with TGF-β for 2 weeks and BGP is added from week 2 of culture. Cartilage mineralisation determines loss of glycosaminoglycans, reduced expression but not protein abundance of collagen II and X, and decreased VEGFA production. Finally, the conditioned medium from mineralised pellets showed a reduced ability to stimulate endothelial cell migration, proliferation and tube formation. The pro-angiogenic potential of transient cartilage is thus stage-dependent, and this aspect must be carefully considered in the design of bone tissue engineering strategies.

Chronic myeloproliferative neoplasms encompass the BCR-ABL1-negative neoplasms polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These are characterized by calreticulin (CALR), myeloproliferative leukemia virus proto-oncogene (MPL) and the tyrosine kinase Janus kinase 2 (JAK2) mutations, eventually establishing a hyperinflammatory tumor microenvironment (TME). Several reports have come to describe how constitutive activation of JAK-STAT and NFκB signaling pathways lead to uncontrolled myeloproliferation and pro-inflammatory cytokines secretion. In such a highly oxidative TME, the balance between Hematopoietic Stem Cells (HSCs) and Mesenchymal Stromal Cells (MSCs) has a crucial role in MPN development. For this reason, we sought to review the current literature concerning the interplay between HSCs and MSCs. The latter have been reported to play an outstanding role in establishing of the typical bone marrow (BM) fibrotic TME as a consequence of the upregulation of different fibrosis-associated genes including PDGF- β upon their exposure to the hyperoxidative TME characterizing MPNs. Therefore, MSCs might turn to be valuable candidates for niche-targeted targeting the synthesis of cytokines and oxidative stress in association with drugs eradicating the hematopoietic clone.

Introduktion: Hyalint brosk består av kondrocyter inbäddade i ett extracellulärt matrix som täcker alla kroppens synoviala leder. I dag är skador på hyalint brosk ett folkhälsoproblem med begränsade behandlingsmöjligheter. Mesenkymala stamceller har i flertalet studier visat sig vara en lovande kandidat för framtida behandling av broskskador, eftersom dessa kan differentiera till broskproducerande kondrocyter. Modern behandling av broskskador innefattar odling av patientens egna kondrocyter eller stamceller för att sedan återföra dem till broskskadan. För att göra detta effektivt behöver optimering av odlingsförhållanden för aktuella celler utföras. Syfte: Att undersöka tillväxt hos kondrocyter samt mesenkymala stromaceller från benmärg i varierande odlingsförhållanden, detta med avseende att utvärdera vilka tillsatser som mest gynnar cellernas viabilitet och proliferation. Material & Metod: Framtagning av kondrocyter och mesenkymala stromaceller från benmärg i denna studie utfördes på överblivet ortopediskt material från en individ. Cellerna kollagenasbehandlades innan de odlades ut i ett standardmedium under tre veckor. Därefter odlades de i närvaro av olika tillsatser under sex dagar. Cellernas viabilitet mättes genom relativ fluorescens med CellTiter-Blue® reagens medan proliferationen mättes genom cellräkning med flödescytometri. Resultat: Det fanns en signifikant skillnad (ANOVA, p, Introduction: Articular cartilage consists of chondrocytes and covers all the synovial joints in the body. Articular cartilage injuries are today a public health issue with limited orthopedic treatments. Mesenchymal stem cells have shown to be a promising candidate for future treatment of articular cartilage injuries due to their many abilities. Modern treatment of cartilage damage involves cultivation of the patient´s own chondrocytes and stem cells. To make this treatment as effective as possible the culture conditions for chondrocytes and stem cells must be optimized. Aim: To study cell growth in chondrocytes and mesenchymal stromal cells from bone marrow in varying culture conditions, this regarding to evaluate the most optimal culture conditions for cell viability and proliferation. Method: Cell extraction in this study was performed on leftover orthopedic material. The cells were treated with collagenase and then cultured in a standard medium for three weeks before they were cultured in presence of various factors for six days. Cell viability was measured by relative fluorescence while proliferation was measured by cell counting. Result: There was a statistically significant difference (ANOVA, p

The investigation of multipotent stem/stromal cells (MSCs) in vitro represents an important basis for translational studies in large animal models. The study’s aim was to examine and compare clinically relevant in vitro properties of equine MSCs, which were isolated from abdominal (abd), retrobulbar (rb) and subcutaneous (sc) adipose tissue by collagenase digestion (ASCs-SVF) and an explant technique (ASCs-EXP). Firstly, we examined proliferation and trilineage differentiation and, secondly, the cardiomyogenic differentiation potential using activin A, bone morphogenetic protein-4 and Dickkopf-1. Fibroblast-like, plastic-adherent ASCs-SVF and ASCs-EXP were obtained from all sources. The proliferation and chondrogenic differentiation potential did not differ significantly between the isolation methods and localizations. However, abd-ASCs-EXP showed the highest adipogenic differentiation potential compared to rb- and sc-ASCs-EXP on day 7 and abd-ASCs-SVF a higher adipogenic potential compared to abd-ASCs-EXP on day 14. Osteogenic differentiation potential was comparable at day 14, but by day 21, abd-ASCs-EXP demonstrated a higher osteogenic potential compared to abd-ASCs-SVF and rb-ASCs-EXP. Cardiomyogenic differentiation could not be achieved. This study provides insight into the proliferation and multilineage differentiation potential of equine ASCs and is expected to provide a basis for future preclinical and clinical studies in horses.

Triple-negative breast cancer (TNBC) is a very aggressive disease even in its early stages and is characterized by a severe prognosis. Neoadjuvant chemotherapy is one of the milestones of treatment, and paclitaxel (PTX) is among the most active drugs used in this setting. However, despite its efficacy, peripheral neuropathy occurs in approximately 20–25% of cases and represents the dose-limiting toxicity of this drug. New deliverable strategies to ameliorate drug delivery and reduce side effects are keenly awaited to improve patients’ outcomes. Mesenchymal stromal cells (MSCs) have recently been demonstrated as promising drug delivery vectors for cancer treatment. The aim of the present preclinical study is to explore the possibility of a cell therapy approach based on the use of MSCs loaded with PTX to treat TNBC-affected patients. For this purpose, we in vitro evaluated the viability, migration and colony formation of two TNBC cell lines, namely, MDA-MB-231 and BT549, treated with MSC-PTX conditioned medium (MSC-CM PTX) in comparison with both CM of MSCs not loaded with PTX (CTRL) and free PTX. We observed stronger inhibitory effects on survival, migration and tumorigenicity for MSC-CM PTX than for CTRL and free PTX in TNBC cell lines. Further studies will provide more information about activity and potentially open the possibility of using this new drug delivery vector in the context of a clinical study.

Bone regenerative medicine is a clinical approach combining live osteoblast progenitors, such as mesenchymal stromal cells (MSCs), with a biocompatible scaffold that can integrate into host bone tissue and restore its structural integrity. Over the last few years, many tissue engineering strategies have been developed and thoroughly investigated; however, limited approaches have been translated to clinical application. Consequently, the development and clinical validation of regenerative approaches remain a centerpiece of investigational efforts towards the clinical translation of advanced bioengineered scaffolds. The aim of this review was to identify the latest clinical trials related to the use of scaffolds with or without MSCs to regenerate bone defects. A revision of the literature was performed in PubMed, Embase, and Clinicaltrials.gov from 2018 up to 2023. Nine clinical trials were analyzed according to the inclusion criteria: six presented in the literature and three reported in Clinicaltrials.gov. Data were extracted covering background trial information. Six of the clinical trials added cells to scaffolds, while three used scaffolds alone. The majority of scaffolds were composed of calcium phosphate ceramic alone, such as β-tricalcium phosphate (TCP) (two clinical trials), biphasic calcium phosphate bioceramic granules (three clinical trials), and anorganic bovine bone (two clinical trials), while bone marrow was the primary source of the MSCs (five clinical trials). The MSC expansion was performed in GMP facilities, using human platelet lysate (PL) as a supplement without osteogenic factors. Only one trial reported minor adverse events. Overall, these findings highlight the importance and efficacy of cell–scaffold constructs in regenerative medicine under different conditions. Despite the encouraging clinical results obtained, further studies are needed to assess their clinical efficacy in treating bone diseases to optimize their application.

Background Poor graft function or graft failure after allogeneic stem cell transplantation is an unmet medical need, in which mesenchymal stromal cells (MSC) constitute an attractive potential therapeutic approach. Hypoxia-inducible factor-1α (HIF-1α) overexpression in MSC (HIF-MSC) potentiates the angiogenic and immunomodulatory properties of these cells, so we hypothesized that co-transplantation of MSC-HIF with CD34+ human cord blood cells would also enhance hematopoietic stem cell engraftment and function both in vitro and in vivo. Methods Human MSC were obtained from dental pulp. Lentiviral overexpression of HIF-1α was performed transducing cells with pWPI-green fluorescent protein (GFP) (MSC WT) or pWPI-HIF-1α-GFP (HIF-MSC) expression vectors. Human cord blood CD34+ cells were co-cultured with MSC WT or HIF-MSC (4:1) for 72 h. Then, viability (Annexin V and 7-AAD), cell cycle, ROS expression and immunophenotyping of key molecules involved in engraftment (CXCR4, CD34, ITGA4, c-KIT) were evaluated by flow cytometry in CD34+ cells. In addition, CD34+ cells clonal expansion was analyzed by clonogenic assays. Finally, in vivo engraftment was measured by flow cytometry 4-weeks after CD34+ cell transplantation with or without intrabone MSC WT or HIF-MSC in NOD/SCID mice. Results We did not observe significant differences in viability, cell cycle and ROS expression between CD34+ cells co-cultured with MSC WT or HIF-MSC. Nevertheless, a significant increase in CD34, CXCR4 and ITGA4 expression (p = 0.009; p = 0.001; p = 0.013, respectively) was observed in CD34+ cells co-cultured with HIF-MSC compared to MSC WT. In addition, CD34+ cells cultured with HIF-MSC displayed a higher CFU-GM clonogenic potential than those cultured with MSC WT (p = 0.048). We also observed a significant increase in CD34+ cells engraftment ability when they were co-transplanted with HIF-MSC compared to CD34+ co-transplanted with MSC WT (p = 0.016) or alone (p = 0.015) in both the injected and contralateral femurs (p = 0.024, p = 0.008 respectively). Conclusions Co-transplantation of human CD34+ cells with HIF-MSC enhances cell engraftment in vivo. This is probably due to the ability of HIF-MSC to increase clonogenic capacity of hematopoietic cells and to induce the expression of adhesion molecules involved in graft survival in the hematopoietic niche.

Summary Mesenchymal stromal cells (MSCs) have shown significant heterogeneity in terms of therapeutic efficacy for inflammatory bowel disease (IBD) treatment, which may be due to an insufficient number of MSCs homing to the damaged tissue of the colon. Engineering MSCs with specific chemokine receptors can enhance the homing ability by lentiviral transduction. However, the unclear specific chemokine profile related to IBD and the safety concerns of viral-based gene delivery limit its application. Thus, a new strategy to modify MSCs to express specific chemokine receptors using mRNA engineering is developed to evaluate the homing ability of MSCs and its therapeutic effects for IBD. We found that CXCL2 and CXCL5 were highly expressed in the inflammatory colon, while MSCs minimally expressed the corresponding receptor CXCR2. Transient expression of CXCR2 in MSC was constructed and exhibited significantly enhanced migration to the inflamed colons, leading to a robust anti-inflammatory effect and high efficacy. Furthermore, the high expression of semaphorins7A on MSCs were found to induce the macrophages to produce IL-10, which may play a critical therapeutic role. This study demonstrated that the specific chemokine receptor CXCR2 mRNA-engineered MSCs not only improves the therapeutic efficacy of IBD but also provides an efficient and safe MSC modification strategy., Graphical abstract, Li et al. demonstrate that mRNA-engineered MSCs with CXCR2 significantly increase their migratory properties to injured organ/tissue in an inflammatory bowel mouse model. This strategy for enhancing targeted stem/progenitor cell homing may provide a potential strategy to improve the efficacy of MSC-based therapeutics and be applicable to clinical patients.