Expansion Of Mesenchymal Stromal Cells Research Articles

Mass Production of Rg1-Loaded Small Extracellular Vesicles Using a 3D Bioreactor System for Enhanced Cardioprotective Efficacy of Doxorubicin-Induced Cardiotoxicity.

Small extracellular vesicles (sEVs) obtained from human umbilical cord mesenchymal stromal cells (MSCs) have shown cardioprotective efficacy in doxorubicin-induced cardiotoxicity (DIC). However, their clinical application is limited due to the low yield and high consumption. This study aims to achieve large-scale production of sEVs using a three-dimensional (3D) bioreactor system. In addition, sEVs were developed to deliver Ginsenoside Rg1 (Rg1), a compound derived from traditional Chinese medicine, Ginseng, that has cardioprotective properties but limited bioavailability, to enhance the treatment of DIC. The 3D bioreactor system with spinner flasks was used to expand human umbilical cord MSCs and collect MSC-conditioned medium. Subsequently, sEVs were isolated from the conditioned medium using differential ultra-centrifugation (dUC). The sEVs were loaded with Ginsenoside Rg1 by electroporation and evaluated for cardioprotective efficacy using Cell Counting Kit-8 (CCK-8) analysis, Annexin V/PI staining and live cell count of H9c2 cells under DIC. Using the 3D bioreactor system with spinner flasks, the expansion of MSCs reached ~600 million, and the production of sEVs was up to 2.2 × 1012 particles in five days with significantly reduced bench work compared to traditional 2D flasks. With the optimized protocol, the Ginsenoside Rg1 loading efficiency of sEVs by electroporation was ~21%, higher than sonication or co-incubation. Moreover, Rg1-loaded sEVs had attenuated DOX-induced cardiotoxicity with reduced apoptosis compared to free Ginsenoside Rg1 or sEVs. The 3D culture system scaled up the production of sEVs, which facilitated the Rg1 delivery and attenuated cardiomyocyte apoptosis, suggesting a potential treatment of DOX-induced cardiotoxicity.

Calcium chloride declotted human platelet lysate promotes the expansion of mesenchymal stromal cells and allows manufacturing of immunomodulatory active extracellular vesicle products

BackgroundMesenchymal stromal cells (MSCs) exert immunomodulatory effects, primarily through released extracellular vesicles (EVs). For the clinical-grade manufacturing of MSC-EV products culture conditions need to support MSC expansion and allow the manufacturing of potent MSC-EV products. Traditionally, MSCs are expanded in fetal bovine serum-supplemented media. However, according to good manufacturing practice (GMP) guidelines the use of animal sera should be avoided. To this end, human platelet lysate (hPL) has been qualified as an animal serum replacement. Although hPL outcompetes animal sera in promoting MSC expansion, hPL typically contains components of the coagulation system that need to be inhibited or removed to avoid coagulation reactions in the cell culture. Commonly, heparin is utilized as an anticoagulant; however, higher concentrations of heparin can negatively impact MSC viability, and conventional concentrations alone do not sufficiently prevent clot formation in prepared media. MethodsTo circumvent unwanted coagulation processes, this study compared various clotting prevention strategies, including different anticoagulants and calcium chloride (CaCl2)-mediated declotting methods, which in combination with heparin addition was found effective. We evaluated the influence of the differently treated hPLs on the proliferation and phenotype of primary bone marrow-derived MSCs and identified the CaCl2-mediated declotting method as the most effective option. To determine whether CaCl2 declotted hPL allows the manufacturing of immunomodulatory MSC-EV products, EVs were prepared from conditioned media of MSCs expanded with either conventional or CaCl2 declotted hPL. In addition to metric analyses, the immunomodulatory potential of resulting MSC-EV products was assessed in a recently established multi-donor mixed lymphocyte reaction assay. Results and ConclusionsOur findings conclusively show that CaCl2-declotted hPLs support the production of immunomodulatory-active MSC-EV products.

The Use of Human Platelet Lysate as a Coating Substance for Adipose-Derived Stem Cell Expansion.

Large-scale production of mesenchymal stromal cells is essential for sufficient therapeutic doses in regenerative medicine. However, long-term cultivation encounters limited cell growth and cellular aging. Therefore, an alternative cell culture approach that promotes proliferation and attenuates cell senescence is required. Human platelet lysate (HPL) is a potent supplement for in vitro cell expansion. Applying HPL as a coating material can potentially improve mesenchymal stromal cell cultures. To examine the capacity of HPL, it was used to pre-coat a tissue culture plate for in vitro adipose-derived mesenchymal stromal cell expansion. Alterations in biological features of adipose-derived stem cells (ADSCs) were investigated, including cell adhesion assays, cell proliferation, population doubling time, and cellular senescence. ADSCs cultured on HPL-coated plates significantly increased cell adhesion rate, shortened population doubling time, and stimulated cell growth. The senescent cells were significantly decreased in ADSCs cultured in an HPL-coated plate, and the expression levels of senescence-associated genes, including p16, p21, and p53, were downregulated. Furthermore, Western blotting analysis revealed that HPL was enriched with fibronectin and vitronectin, essential cell adhesive proteins. HPL was effectively used as a coating material for ADSC expansions. Cellular cultivation on the HPL coating is an alternative approach for producing mesenchymal stromal cells.

Media matters: culture medium-dependent hypervariable phenotype of mesenchymal stromal cells

BackgroundDespite a long history of investigation and sustained efforts in clinical testing, the number of market authorisations for mesenchymal stromal cell (MSC) therapies remains limited, with none approved by the United States Food and Drug Administration. Several barriers are impeding the clinical progression of MSC therapies, to the forefront of these is a lack of standardised manufacturing protocols which is further compounded by an absence of biologically meaningful characterisation and 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, among 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.MethodsThis work analysed the influence of five widely used culture media formulations on the colony-forming potential, proliferation kinetics, trilineage differentiation potential and immunomodulatory potential of human bone marrow-derived MSCs (BM-MSCs). The surface marker expression profiles were also characterised using a high-content flow cytometry screening panel of 243 markers.ResultsSignificant differences in the biological attributes of BM-MSCs including clonogenicity, proliferation, differentiation propensity and immunomodulatory capacity were revealed in response to the composition of the culture medium. Despite their biological differences, all cell preparations uniformly and strongly expressed the standard positive markers proposed for BM-MSCs: CD73, CD90 and CD105. Immunophenotypic profiling revealed that the culture medium also had a significant influence on the surface proteome, with one-third of tested markers exhibiting variable expression profiles. Principal component analysis demonstrated that BM-MSCs isolated and expanded in a proprietary xeno- and serum-free medium displayed the most consistent cell phenotypes with little variability between donors compared to platelet lysate and foetal bovine serum-containing media.ConclusionsThese data suggest that media composition has a highly significant impact on the biological attributes of MSCs, but standard surface marker tests conceal these differences. The results indicate a need for (1) standardised approaches to manufacturing, with an essential focus on defined media and (2) new biologically relevant tests for MSC characterisation and product release.

Local manufacturing processes contribute to variability in human mesenchymal stromal cell expansion while growth media supplements contribute to variability in gene expression and cell function: a Biomedical Excellence for Safer Transfusion (BEST) collaborative study

Background aimsCulture-derived mesenchymal stromal cells (MSCs) exhibit variable characteristics when manufactured using different methods, source material and culture media. The purpose of this multicenter study was to assess the impact on MSC expansion, gene expression and other characteristics when different laboratories expanded MSCs from cultures initiated with bone marrow−MSC aliquots derived from the same donor source material yet with different growth media. MethodsEight centers expanded MSCs using four human platelet lysate (HPL) and one fetal bovine serum (FBS) products as media supplements. The expanded cells were taken through two passages then assessed for cell count, viability, doubling time, immunophenotype, cell function, immunosuppression and gene expression. Results were analyzed by growth media and by center. ResultsCenter methodologies varied by their local seeding density, feeding regimen, inoculation density, base media and other growth media features (antibiotics, glutamine, serum). Doubling times were more dependent on center than on media supplements. Two centers had appropriate immunophenotyping showing all MSC cultures were positive for CD105, CD73, CD90 and negative for CD34, CD45, CD14, HLA-DR. MSCs cultured in media supplemented with FBS compared with HPL featured greater T-cell inhibition potential. Gene expression analysis showed greater impact of the type of media supplement (HPL versus FBS) than the manufacturing center. Specifically, nine genes were decreased in expression and six increased when combining the four HPL-grown MSCs versus FBS (false discovery rate [FDR] <0.01), however, without significant difference between different sources of HPL (FDR <0.01). ConclusionsLocal manufacturing process plays a critical role in MSC expansion while growth media may influence function and gene expression. All HPL and FBS products supported cell growth.

Mapping the Cellular Biogeography of Human Bone Marrow Niches Using Single-Cell Transcriptomics and Proteomic Imaging

Introduction Rare non-hematopoietic cells in the bone marrow make essential contributions to hematopoiesis. Despite tremendous progress in understanding the mouse bone marrow microenvironment, the precise identity of human non-hematopoietic bone marrow cells and their spatial organization remain largely uncharacterized. Here, we performed single cell transcriptomics and spatial proteomics, assembling the first comprehensive atlas of human bone marrow cellular composition and organization. Methods Using fresh femoral head samples obtained from orthopedic hip replacement surgeries, we first performed single-cell RNA sequencing (scRNA-Seq) to create a comprehensive human bone marrow atlas profiling 29,325 enriched non-hematopoietic cells as well as 53,417 hematopoietic cells from enzymatically digested femoral heads from 12 individuals. We next employed Co-Detection by Indexing (CODEX) multiplexed imaging of 18 bone marrow samples, including both healthy and acute myeloid leukemia (AML) samples, to spatially profile over one million single cells with a novel 53-antibody panel. Results Our transcriptional analysis revealed nine distinct non-hematopoietic bone marrow cell types including mesenchymal stromal cell (MSC) subsets, osteolineage cells, vascular smooth muscle cells, and both sinusoidal and arterial endothelial cells (Figure 1A). We found that MSCs were heterogeneous and hierarchically organized. One specific cell cluster, which we termed Fibro-MSCs, corresponded to previous descriptions of mesenchymal stem cells and was both computationally and functionally shown to be the most stem/progenitor-like. Next, we performed computational cellular communication analysis which revealed that two MSC subsets, Adipo and THY1+ MSCs, provided the majority of canonical hematopoietic supportive factors such as CXCL12, KITLG, IL7, and PTN. Next, we used CODEX to systematically study spatial relationships between these cells. Consistent with the scRNA-Seq cellular communication analysis, we found that Adipo and THY1+ MSCs were in the bone marrow interacting with hematopoietic cells, while osteolineage cells and Fibro-MSCs were found within the trabecular bone region. We then performed unsupervised neighborhood analysis to analyze the distinct niches of the bone marrow. We discovered a relatively hyperoxygenated arterio-endosteal niche for early myelopoiesis, where early myeloid progenitors were characterized by very low levels of HIF1α, in contrast to HIF1α hi neutrophils which were found largely residing near sinusoids. We also employed point pattern statistics and permutation tests to analyze the proximity of cell types to manually identified structures in the bone marrow. This analysis revealed that CD34+ hematopoietic stem and progenitor cells (HSPCs) were frequently contacting adipocytes (Figure 1B), but not sinusoids or bone. Collectively, these results led us to propose a data-driven working model for how human myelopoiesis is spatially organized, with the earliest myeloid progenitors being specified from HSPCs near adipocytes, then migrating to the peri-arteriolar/peri-endosteal early myeloid progenitor niche, and then finally localizing near sinusoids which facilitates bone marrow egress as they mature to neutrophils. To evaluate if our CODEX atlas could be used to study disease states, we mapped new bone marrow images from acute myeloid leukemia (AML) patients and controls to our reference. We used machine learning to identify true mutant cells based on NPM1c expression patterns. We discovered Adipo and THY1+ MSC expansion in AML patients compared to controls, with expanded MSCs spatially associated with leukemic blasts. Our results highlight the potential of our CODEX atlas to contextualize new datasets and study bone marrow organization in disease states, and provide further rationale to study MSC-AML blast interactions. Conclusions Taken together, our scRNA-Seq and CODEX data act in concert, demonstrating the diversity of non-hematopoietic stromal elements, establishing the source of hematopoietic cytokines, and defining the spatial organization in healthy human bone marrow. We envision our multiomic, spatially resolved atlas of human bone marrow will serve as a critical resource for future study of the bone marrow microenvironment in both healthy and disease states.

Bioprocess development for cord blood mesenchymal stromal cells on microcarriers in Vertical-Wheel bioreactors.

Equine mesenchymal stromal cells (MSCs) have been found to be beneficial for the treatment of many ailments, including orthopedic injuries, due to their superior differentiation potential and immunomodulating properties. Cell therapies require large cell numbers, which are not efficiently generated using conventional static expansion methods. Expansion of equine cord blood-derived MSCs (eCB-MSCs) in bioreactors, using microcarriers as an attachment surface, has the potential to generate large numbers of cells with increased reproducibility and homogeneity compared with static T-flask expansion. This study investigated the development of an expansion process using Vertical-Wheel (VW) bioreactors, a single-use bioreactor technology that incorporates a wheel instead of an impeller. Initially, microcarriers were screened at small scale to assess eCB-MSC attachment and growth and then in bioreactors to assess cell expansion and harvesting. The effect of different donors, serial passaging, and batch versus fed batch were all examined in 0.1 L VW bioreactors. The use of VW bioreactors with an appropriate microcarrier was shown to be able to produce cell densities of up to 1E6 cells/mL, while maintaining cell phenotype and functionality, thus demonstrating great potential for the use of these bioreactors to produce large cell numbers for cell therapies.

Effect of Expansion Media on Functional Characteristics of Bone Marrow-Derived Mesenchymal Stromal Cells.

The therapeutic efficacy of mesenchymal stromal cells (MSCs) has been shown to rely on their immunomodulatory and regenerative properties. In order to obtain sufficient numbers of cells for clinical applications, MSCs have to be expanded ex vivo. Expansion media with xenogeneic-free (XF) growth-promoting supplements like human platelet lysate (PL) or serum- and xenogeneic-free (SF/XF) formulations have been established as safe and efficient, and both groups provide different beneficial qualities. In this study, MSCs were expanded in XF or SF/XF media as well as in mixtures thereof. MSCs cultured in these media were analyzed for phenotypic and functional properties. MSC expansion was optimal with SF/XF conditions when PL was present. Metabolic patterns, consumption of growth factors, and secretome of MSCs differed depending on the type and concentration of supplement. The lactate per glucose yield increased along with a higher proportion of PL. Many factors in the supernatant of cultured MSCs showed distinct patterns depending on the supplement (e.g., FGF-2, TGFβ, and insulin only in PL-expanded MSC, and leptin, sCD40L PDGF-AA only in SF/XF-expanded MSC). This also resulted in changes in cell characteristics like migratory potential. These findings support current approaches where growth media may be utilized for priming MSCs for specific therapeutic applications.

Releasing YAP dysfunction-caused replicative toxicity rejuvenates mesenchymal stem cells.

Hippo-independent YAP dysfunction has been demonstrated to cause chronological aging of stromal cells by impairing the integrity of nuclear envelope (NE). In parallel with this report, we uncover that YAP activity also controls another type of cellular senescence, the replicative senescence in in vitro expansion of mesenchymal stromal cells (MSCs), but this event is Hippo phosphorylation-dependent, and there exist another NE integrity-independent downstream mechanisms of YAP. Specifically, Hippo phosphorylation causes reduced nuclear/active YAP and then decreases the level of YAP protein in the proceeding of replicative senescence. YAP/TEAD governs RRM2 expression to release replicative toxicity (RT) via licensing G1/S transition. Besides, YAP controls the core transcriptomics of RT to delay the onset of genome instability and enhances DNA damage response/repair. Hippo-off mutations of YAP (YAPS127A/S381A ) satisfactorily release RT via maintaining cell cycle and reducing genome instability, finally rejuvenating MSCs and restoring their regenerative capabilities without risks of tumorigenesis.

Bone Regeneration Using Mesenchymal Stromal Cells and Biocompatible Scaffolds: A Concise Review of the Current Clinical Trials.

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.

Macromolecular crowding in animal component-free, xeno-free and foetal bovine serum media for human bone marrow mesenchymal stromal cell expansion and differentiation.

Background: Cell culture media containing undefined animal-derived components and prolonged in vitro culture periods in the absence of native extracellular matrix result in phenotypic drift of human bone marrow stromal cells (hBMSCs). Methods: Herein, we assessed whether animal component-free (ACF) or xeno-free (XF) media formulations maintain hBMSC phenotypic characteristics more effectively than foetal bovine serum (FBS)-based media. In addition, we assessed whether tissue-specific extracellular matrix, induced via macromolecular crowding (MMC) during expansion and/or differentiation, can more tightly control hBMSC fate. Results: Cells expanded in animal component-free media showed overall the highest phenotype maintenance, as judged by cluster of differentiation expression analysis. Contrary to FBS media, ACF and XF media increased cellularity over time in culture, as measured by total DNA concentration. While MMC with Ficoll™ increased collagen deposition of cells in FBS media, FBS media induced significantly lower collagen synthesis and/or deposition than the ACF and XF media. Cells expanded in FBS media showed higher adipogenic differentiation than ACF and XF media, which was augmented by MMC with Ficoll™ during expansion. Similarly, Ficoll™ crowding also increased chondrogenic differentiation. Of note, donor-to-donor variability was observed for collagen type I deposition and trilineage differentiation capacity of hBMSCs. Conclusion: Collectively, our data indicate that appropriate screening of donors, media and supplements, in this case MMC agent, should be conducted for the development of clinically relevant hBMSC medicines.

Nanotopography reveals metabolites that maintain the immunomodulatory phenotype of mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are multipotent progenitor cells that are of considerable clinical potential in transplantation and anti-inflammatory therapies due to their capacity for tissue repair and immunomodulation. However, MSCs rapidly differentiate once in culture, making their large-scale expansion for use in immunomodulatory therapies challenging. Although the differentiation mechanisms of MSCs have been extensively investigated using materials, little is known about how materials can influence paracrine activities of MSCs. Here, we show that nanotopography can control the immunomodulatory capacity of MSCs through decreased intracellular tension and increasing oxidative glycolysis. We use nanotopography to identify bioactive metabolites that modulate intracellular tension, growth and immunomodulatory phenotype of MSCs in standard culture and during larger scale cell manufacture. Our findings demonstrate an effective route to support large-scale expansion of functional MSCs for therapeutic purposes.

Glucose Metabolism: Optimizing Regenerative Functionalities of Mesenchymal Stromal Cells Postimplantation.

Mesenchymal stromal cells (MSCs) are considered promising candidates for regenerative medicine applications. Their clinical performance postimplantation, however, has been disappointing. This lack of therapeutic efficacy is most likely due to suboptimal formulations of MSC-containing material constructs. Tissue engineers, therefore, have developed strategies addressing/incorporating optimized cell, microenvironmental, biochemical, and biophysical cues/stimuli to enhance MSC-containing construct performance. Such approaches have had limited success because they overlooked that maintenance of MSC viability after implantation for a sufficient time is necessary for MSCs to develop their regenerative functionalities fully. Following a brief overview of glucose metabolism and regulation in MSCs, the present literature review includes recent pertinent findings that challenge old paradigms and notions. We hereby report that glucose is the primary energy substrate for MSCs, provides precursors for biomass generation, and regulates MSC functions, including proliferation and immunosuppressive properties. More importantly, glucose metabolism is central in controlling in vitro MSC expansion, in vivo MSC viability, and MSC-mediated angiogenesis postimplantation when addressing MSC-based therapies. Meanwhile, in silico models are highlighted for predicting the glucose needs of MSCs in specific regenerative medicine settings, which will eventually enable tissue engineers to design viable and potent tissue constructs. This new knowledge should be incorporated into developing novel effective MSC-based therapies. Impact statement The clinical use of mesenchymal stromal cells (MSCs) has been unsatisfactory due to the inability of MSCs to survive and be functional after implantation for sufficient periods to mediate directly or indirectly a successful regenerative tissue response. The present review summarizes the endeavors in the past, but, most importantly, reports the latest findings that elucidate underlying mechanisms and identify glucose metabolism as the crucial parameter in MSC survival and the subsequent functions pertinent to new tissue formation of importance in tissue regeneration applications. These latest findings justify further basic research and the impetus for developing new strategies to improve the modalities and efficacy of MSC-based therapies.

A controllable gelatin-based microcarriers fabrication system for the whole procedures of MSCs amplification and tissue engineering.

Biopolymer microbeads present substantial benefits for cell expansion, tissue engineering, and drug release applications. However, a fabrication system capable of producing homogeneous microspheres with high precision and controllability for cell proliferation, passaging, harvesting and downstream application is limited. Therefore, we developed a co-flow microfluidics-based system for the generation of uniform and size-controllable gelatin-based microcarriers (GMs) for mesenchymal stromal cells (MSCs) expansion and tissue engineering. Our evaluation of GMs revealed superior homogeneity and efficiency of cellular attachment, expansion and harvest, and MSCs expanded on GMs exhibited high viability while retaining differentiation multipotency. Optimization of passaging and harvesting protocols was achieved through the addition of blank GMs and treatment with collagenase, respectively. Furthermore, we demonstrated that MSC-loaded GMs were printable and could serve as building blocks for tissue regeneration scaffolds. These results suggested that our platform held promise for the fabrication of uniform GMs with downstream application of MSC culture, expansion and tissue engineering.

Xeno-free cultured mesenchymal stromal cells release extracellular vesicles with a "therapeutic" miRNA cargo ameliorating cartilage inflammation in vitro.

Rationale: Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) emerged as an innovative strategy for the treatment of chronic disorders such as osteoarthritis (OA). Biological activity of EVs is generally driven by their cargo, which might be influenced by microenvironment. Therefore, pre-conditioning strategies, including modifications in culture conditions or oxygen tension could directly impact on MSCs paracrine activity. In this study we selected an appropriate preconditioning system to induce cells to perform the most suitable therapeutic response by EV-encapsulated bioactive factors. Methods: A xeno-free supplement (XFS) was used for isolation and expansion of MSCs and compared to conventional fetal bovine serum (FBS) culture. Bone Marrow-derived MSCs (BMSCs) were pre-conditioned under normoxia (20% O2) or under hypoxia (1% O2) and EVs production was evaluated. Anti-OA activity was evaluated by using an in vitro inflammatory model. miRNA content was also explored, to select putative miRNA that could be involved in a biological function. Results: Modulation of IL-6, IL-8, COX-2 and PGE2 was evaluated on hACs simultaneously treated with IL-1α and BMSC-derived EVs. FBS-sEVs exerted a blunt inhibitory effect, while a strong anti-inflammatory outcome was achieved by XFS-sEVs. Interestingly, in both cases hypoxia pre-conditioning allowed to increase EVs effectiveness. Analysis of miRNA content showed the upregulation in XFS-hBMSC-derived EVs of miRNA known to have a chondroprotective role, such as let-7b-5p, miR-17, miR-145, miR-21-5p, miR-214-3p, miR-30b-5p, miR-30c-5p. Activated pathways and target genes were investigated in silico and upregulated miRNAs functionally validated in target cells. MiR-145 and miR-214 were found to protect chondrocytes from IL-1α-induced inflammation and to reduce production of pro-inflammatory cytokines. Conclusions: XFS medium was found to be suitable for isolation and expansion of MSCs, secreting EVs with a therapeutic cargo. The application of cells cultured exclusively in XFS overcomes issues of safety associated with serum-containing media and makes ready-to-use clinical therapies more accessible.

Cross-Talk between Hematopoietic Cells and Fibroblast Subsets Drives Inflammation and Remodelling of the Bone Marrow Microenvironment in Myeloproliferative Neoplasms

Introduction: In a proportion of patients with myeloproliferative neoplasms (MPNs), severe remodelling of the bone marrow microenvironment develops, with myelofibrosis (MF) and osteosclerosis leading to cytopenias and poor survival. Mesenchymal stromal cells (MSCs) have been implicated in MF pathogenesis (Leimkühler, Cell Stem Cell, 2021), including Nestin+ (Arranz, Nature, 2014), Gli1+ (Schneider, Cell Stem Cell, 2017) and Leptin-receptor+ MSCs (Decker, Nat Cell Bio, 2017). However, little is known about the cellular subtypes and transcriptional states of fibroblast subtypes in MF, and the key receptor-ligand (R-L) interactions that lead to their activation. Our aim was to build a fully comprehensive atlas of MF bone marrow and clarify the pathological interactions between MPN cells and their stromal environment to determine the key drivers of inflammation and fibrosis. Methods: We performed single cell RNA sequencing of mononuclear cells, hematopoietic stem/progenitor cells (HSPCs), CD41+ cells and CD45- Lin- Ter119- CD71low/- stromal cells from MF (MPLW515L) mice and controls, as well as CD45- Lin- CD235ab- CD71low/- stromal cells from MPN patients. Targets were validated using a novel human bone marrow organoid platform(Khan et al, bioRxiv, 2022) and by analysis of bone marrow biopsies from MPN patients and controls. Results: More than 75,000 cells were analysed from control and MF mice, capturing 42,319 hematopoietic cells (including HSPCs, myeloid, lymphoid, megakaryocytes) and 34,969 stromal cells, including fibroblasts, MSCs, osteoblasts, chondrocytes, pericytes, arterial and sinusoidal endothelial cells (Fig.1A). Erythroid, neutrophil, megakaryocytes and also eosinophil-basophil-mast (EBM) cells were increased in abundance in MF mice, together with a ~4 - 8 fold expansion of MSCs and a more modest increase in overall abundance of fibroblasts. Extracellular matrix factors were expressed mainly by stromal cells (MSCs, fibroblasts) but also by EBM cells and neutrophils. As reported (Leimkühler, Cell Stem Cell, 2021), expression of hematopoiesis support factors by MSCs was reduced in MF. However, prior studies have not captured fibroblast subtypes. We found that the reduction in niche support from MSCs was compensated by a significant increase in hematopoiesis support factors from fibroblasts and EBM cells. Despite minimal increase in fibroblast cells overall, a subset of inflammatory fibroblasts (iFib) with high metabolic and glycolytic activity, TNFα signaling via NF-κB and TGFβ signaling was dramatically expanded in the MF mice, and also detectable in stromal cells from MPN patients. This atlas enabled detailed study of aberrant cross-talk between hematopoietic and stromal compartments. Overall, receptor-ligand (R-L) interactions were 20% higher in MF mice due to increased signaling from megakaryocytes, EBM, MSC and iFib cells, implicating these four cellular subsets as key mediators of pathogenic cross-talk. R-L interaction analyses predicted increased TNFα signaling (TNFRSF1A → TNF) from EBM to both MSC and iFib, and increased TGFβ signaling (TGFBR2 → TGFB1) between EBM and megakaryocytes to MSC and fibroblast subtypes, including iFib. The expanded subsets of megakaryocytes and EBM cells were noted to express notably high levels of galectins LGALS1 and LGALS3, encoding soluble β-galactoside-binding proteins associated with inflammation and tumorigenesis, and galectin signaling via predicted interacting partners was significantly increased in MF. High LGALS1 correlated with significantly shorter survival in patients with myeloid malignancies in the Cancer Genome Atlas myeloid leukemia cohort. Inhibition of galectin-1 with OTX008 significantly reduced TGFβ-induced collagen 1 and αSMA expression by human bone marrow stromal cells in 2D cultures as well as in human iPSC-derived bone marrow organoids, and bone marrow biopsies from a cohort of MF and non-fibrotic ET/PV patients showed a striking association of galectin-1 with fibrosis (Fig.1B). Conclusions: We present a roadmap of the cellular and molecular landscape of normal and myelofibrotic bone marrow. We identify two cellular subsets (EBM and iFib) whose role as mediators of the inflammatory microenvironment in MF was previously unappreciated, and propose galectin-1 as a novel biomarker and potential mediator of fibrosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MSC and HSPC Coculture: Mimicking Ex Vivo Bone Marrow Niche.

Mesenchymal stromal cells (MSCs) are the crucial component of the hematopoietic stem and progenitor cell (HSPC) niche in the bone marrow. Therefore, an ex vivo culture system that recapitulates the marrow microenvironment is important to understanding the niche's regulatory role on HSPC function and improving ex vivo HSPC expansion for clinical transplantation. Herein, a procedure for ex vivo expansion of MSCs from human bone marrow cells and their identification and characterization is described. In addition, a protocol for MSC and HSPC coculture assay is presented. This MSC-HSPC coculture assay can be used for ex vivo expansion of HSPC. Furthermore, this assay is also useful for qualitative analysis of MSCs capable of supporting hematopoiesis.

Genetic profiling of human bone marrow mesenchymal stromal cells after in vitro expansion in clinical grade human platelet lysate.

Mesenchymal stromal cells (MSCs) are non-hematopoietic cells that have a broad therapeutic potential. To obtain sufficient cells for clinical application, they must be expanded ex vivo. In the initial expansion protocols described, fetal calf serum (FCS) was used as the reference growth supplement, but more recently different groups started to replace FCS with platelet lysate (PL). We investigated in this study the impact of the culture supplement on gene expression of MSCs. Human bone marrow derived MSCs were expanded in vitro in FCS and PL supplemented medium. We found that MSCs expanded in PL-containing medium (PL-MSCs) express typical MSC immunomorphological features and can migrate, as their counterparts expanded in FCS-containing medium, through a layer of endothelial cells in vitro. Additionally, they show an increased proliferation rate compared to MSCs expanded in FCS medium (FCS-MSCs). RNA sequencing performed for MSCs cultured in both types of expansion medium revealed a large impact of the choice of growth supplement on gene expression: 1974 genes were at least twofold up- or downregulated. We focused on impact of genes involved in apoptosis and senescence. Our data showed that PL-MSCs express more anti-apoptotic genes and FCS-MSCs more pro-apoptotic genes. FCS-MSCs showed upregulation of senescence-related genes after four passages whereas this was rarer in PL-MSCs at the same timepoint. Since PL-MSCs show higher proliferation rates and anti-apoptotic gene expression, they might acquire features that predispose them to malignant transformation. We screened 10 MSC samples expanded in PL-based medium for the presence of tumor-associated genetic variants using a 165 gene panel and detected only 21 different genetic variants. According to our analysis, none of these were established pathogenic mutations. Our data show that differences in culture conditions such as growth supplement have a significant impact on the gene expression profile of MSCs and favor the use of PL over FCS for expansion of MSCs.

Abstract 18 Developing a Potency Assay for Cord Tissue MSCs

IntroductionThe potency of mesenchymal stromal cells (MSCs) can be assessed by their ability to suppress proliferation of third party stimulated peripheral blood T cells. This correlates with decreased cellular production of IL-2Ra.ObjectiveWe optimized a potency assay for human cord tissue MSCs (hCT-MSC) in clinical trials for subjects with neuroinflammation. Originally, this T cell suppression assay required radioactive tritiated thymidine, rendering the assay difficult to use in a quality control laboratory for product release. Accordingly, we are developing a modified assay that eliminates the tritiated thymidine and instead tests for production of IL-2Rα in harvested supernatants.MethodsThe modified assay utilizes freshly thawed hCT-MSCs plated in a 96-well Corning CellBIND plate in Fuji Prime-XV XSFM MSC expansion medium at three concentrations. The hCT-MSCs are incubated 1-6 days at 37°C with humidified 5% CO2 until 80%-100% confluent. At this point, three different, previously qualified, normal control peripheral blood mononuclear cells (PBMCs) are suspended in RPMI 1640 with HEPES medium and 10% fetal bovine serum (FBS) with and without Dynabead (Human T-Activator CD3/CD28). After 4 additional days in culture, 75 µL of supernatant is removed and frozen at –80°C for future assaying of IL-10, TNFα, IFNg, and IL-2Ra using the Ella (Bio-Techne). IL-2Ra was determined to have the best correlation with the original method of suppression of proliferation. In the original radioactivity method after 4 days, the cells were pulsed with tritiated thymidine, harvested 6-10 hours after pulsing, and counted in the MicroBeta2 counter.ResultsSeventeen hCT-MSC lines were then tested in both assays. The passing criteria was >70% suppression in the radioactivity assays or >70% decrease in IL-2Ra production in the Ella assay. All 17 hCT-MSC lots passed in the radioactivity-based assay, while 16/17 lines passed using the IL-2Rα assay. Subsequently, the number of PBMCs plated per well and the number of days the PBMCs were in culture were optimized to maximize production of IL-2Rα.DiscussionWe developed a potency assay for MSCs that eliminates the use of radioactive reagents and usable as a release assay in manufacturing of hCT-MSC.

Msx1+ stem cells recruited by bioactive tissue engineering graft for bone regeneration

Critical-sized bone defects often lead to non-union and full-thickness defects of the calvarium specifically still present reconstructive challenges. In this study, we show that neurotrophic supplements induce robust in vitro expansion of mesenchymal stromal cells, and in situ transplantation of neurotrophic supplements-incorporated 3D-printed hydrogel grafts promote full-thickness regeneration of critical-sized bone defects. Single-cell RNA sequencing analysis reveals that a unique atlas of in situ stem/progenitor cells is generated during the calvarial bone healing in vivo. Notably, we find a local expansion of resident Msx1+ skeletal stem cells after transplantation of the in situ cell culture system. Moreover, the enhanced calvarial bone regeneration is accompanied by an increased endochondral ossification that closely correlates to the Msx1+ skeletal stem cells. Our findings illustrate the time-saving and regenerative efficacy of in situ cell culture systems targeting major cell subpopulations in vivo for rapid bone tissue regeneration.