Bone Marrow hMSCs Research Articles

Single-cell RNA-seq of out-of-thaw mesenchymal stromal cells shows tissue-of-origin differences and inter-donor cell-cycle variations

BackgroundHuman Mesenchymal stromal cells (hMSCs) from various tissue sources are widely investigated in clinical trials. These MSCs are often administered to patients immediately after thawing the cryopreserved product (out-of-thaw), yet little is known about the single-cell transcriptomic landscape and tissue-specific differences of out-of-thaw human MSCs.Methods13 hMSC samples derived from 10 “healthy” donors were used to assess donor variability and tissue-of-origin differences in single-cell gene expression profiles. hMSCs derived and expanded from the bone marrow (BM) or cord tissue (CT) underwent controlled-rate freezing for 24 h. Cells were then transferred to the vapor phase of liquid nitrogen for cryopreservation. hMSCs cryopreserved for at least one week, were characterized immediately after thawing using a droplet-based single-cell RNA sequencing method. Data analysis was performed with SC3 and SEURAT pipelines followed by gene ontology analysis.ResultsscRNA-seq analysis of the hMSCs revealed two major clusters of donor profiles, which differ in immune-signaling, cell surface properties, abundance of cell-cycle related transcripts, and metabolic pathways of interest. Within-sample transcriptomic heterogeneity is low. We identified numerous differentially expressed genes (DEGs) that are associated with various cellular functions, such as cytokine signaling, cell proliferation, cell adhesion, cholesterol/steroid biosynthesis, and regulation of apoptosis. Gene-set enrichment analyses indicated different functional pathways in BM vs. CT hMSCs. In addition, MSC-batches showed significant variations in cell cycle status, suggesting different proliferative vs. immunomodulatory potential. Several potential transcript-markers for tissue source differences were identified for further investigation in future studies. In functional assays, both BM and CT MSCs suppressed macrophage TNFα secretion upon interferon stimulation. However, differences between donors, tissue-of-origin, and cell cycle are evident in both TNF suppression and cytokine secretion.ConclusionsThis study shows that donor differences in hMSC transcriptome are minor relative to the intrinsic differences in tissue-of-origin. hMSCs with different transcriptomic profiles showed potential differences in functional characteristics. These findings contribute to our understanding of tissue origin-based differences in out-of-thaw therapeutic hMSC products and assist in the identification of cells with immune-regulatory or survival potential from a heterogeneous MSC population. Our results form the basis of future studies in correlating single-cell transcriptomic markers with immunomodulatory functions.

EFFECT OF HUMAN PLATELET LYSATE ON HUMAN MESENCHYMAIS STEM CELL SUPPLEMENTATION AND APPLICATION IN ACUTE NEURAL INJURY MODEL: STAB WOUND

Traumatic brain injury(TBI) affects millions of people around the world resulting in acute or chronic motor deficiencies.At long-term, TBI causes cognitive/behavioral commitment impacting severely in the life quality. Currently, treatments for TBI are still limited, but mesenchymal stem cells(hMSCs) therapy, due to their plastic, neuroregenerative and immunoregulatory abilities are considered a promisor strategy. Objective: This study we intend to evaluate the impact of supplementation with Human Platelet Lysate(hPL) on the cultivation of hMSCs and its application in an acute neuronal traumatic experimental model Stab Wound(SW). Methodology: Bone marrow hMSCs were cultivated under two conditions: i)with 10% of Bovine Fetal Serum (FBS_hMSCs) or ii)with 10% of highly concentrated hPL(hPL_MSCs).Subsequently both cells population were characterized "in vitro" according to immunophenotype panel, markers of proliferation and oxidative stress(Ki-67 and JC-1).The neurodifferentiation of hMSCs was evaluated by immunofluorescence(β Tubulin-3) and RT-PCR(NeuN,Nestin,GFAP,Iba,NeuroG2 and NeuroD1 genes).The therapeutic potential was investigated "in vivo" using the SW model and histopathological analysis were performed(Iba and DCX).Results: Both hMSCs subsets were fibroblastoid and presented the characteristic MSCs phenotype profile(negative for CD14,CD31,CD45, and positive for CD73,CD90,CD105,CD117,CD133/2,HLA-DR),however hPL_MSCs showed a reduction in the expression of PDL-1 and PDL-2.Along the passages, the hPL_MSCs cultures showed progressively unexpected morphological changes (reduction in the number of nucleolus and cell augmentation/elongation),suggesting a pseudo-neurodifferentiation "in vitro", which was further confirmed by neuronal markers up-regulation.The FBS_hMSCs cultures remained morphologically unaltered. When compared to the FBS_hMSCs cultures, the hPL_MSCs also presented lower cellular stress with a reduced proliferative potential. We analyzed the brain damage tissues infused with both cells, the neuroprotective potential (DCX+ cells) was quite similar, however the neuronal sections treated with hPL_MSCs showed low presence of Iba+ inflammatory cells at the injury site. Conclusion: Preliminary, our results suggest that hPL_MSCs present classic neuronal characteristics “in vitro” with a neuroregenerative potential “in vivo”, and suggesting that hPL is a promisor inductor of neural-like cells being a potential tool for cellular therapy in TBI context. Traumatic brain injury(TBI) affects millions of people around the world resulting in acute or chronic motor deficiencies.At long-term, TBI causes cognitive/behavioral commitment impacting severely in the life quality. Currently, treatments for TBI are still limited, but mesenchymal stem cells(hMSCs) therapy, due to their plastic, neuroregenerative and immunoregulatory abilities are considered a promisor strategy. Objective: This study we intend to evaluate the impact of supplementation with Human Platelet Lysate(hPL) on the cultivation of hMSCs and its application in an acute neuronal traumatic experimental model Stab Wound(SW). Methodology: Bone marrow hMSCs were cultivated under two conditions: i)with 10% of Bovine Fetal Serum (FBS_hMSCs) or ii)with 10% of highly concentrated hPL(hPL_MSCs).Subsequently both cells population were characterized "in vitro" according to immunophenotype panel, markers of proliferation and oxidative stress(Ki-67 and JC-1).The neurodifferentiation of hMSCs was evaluated by immunofluorescence(β Tubulin-3) and RT-PCR(NeuN,Nestin,GFAP,Iba,NeuroG2 and NeuroD1 genes).The therapeutic potential was investigated "in vivo" using the SW model and histopathological analysis were performed(Iba and DCX).Results: Both hMSCs subsets were fibroblastoid and presented the characteristic MSCs phenotype profile(negative for CD14,CD31,CD45, and positive for CD73,CD90,CD105,CD117,CD133/2,HLA-DR),however hPL_MSCs showed a reduction in the expression of PDL-1 and PDL-2.Along the passages, the hPL_MSCs cultures showed progressively unexpected morphological changes (reduction in the number of nucleolus and cell augmentation/elongation),suggesting a pseudo-neurodifferentiation "in vitro", which was further confirmed by neuronal markers up-regulation.The FBS_hMSCs cultures remained morphologically unaltered. When compared to the FBS_hMSCs cultures, the hPL_MSCs also presented lower cellular stress with a reduced proliferative potential. We analyzed the brain damage tissues infused with both cells, the neuroprotective potential (DCX+ cells) was quite similar, however the neuronal sections treated with hPL_MSCs showed low presence of Iba+ inflammatory cells at the injury site. Conclusion: Preliminary, our results suggest that hPL_MSCs present classic neuronal characteristics “in vitro” with a neuroregenerative potential “in vivo”, and suggesting that hPL is a promisor inductor of neural-like cells being a potential tool for cellular therapy in TBI context.

Human Cardiac Mesenchymal Stem Cells Remodel in Disease and Can Regulate Arrhythmia Substrates.

The mesenchymal stem cell (MSC), known to remodel in disease and have an extensive secretome, has recently been isolated from the human heart. However, the effects of normal and diseased cardiac MSCs on myocyte electrophysiology remain unclear. We hypothesize that in disease the inflammatory secretome of cardiac human MSCs (hMSCs) remodels and can regulate arrhythmia substrates. hMSCs were isolated from patients with or without heart failure from tissue attached to extracted device leads and from samples taken from explanted/donor hearts. Failing hMSCs or nonfailing hMSCs were cocultured with normal human cardiac myocytes derived from induced pluripotent stem cells. Using fluorescent indicators, action potential duration, Ca2+ alternans, and spontaneous calcium release (SCR) incidence were determined. Failing and nonfailing hMSCs from both sources exhibited similar trilineage differentiation potential and cell surface marker expression as bone marrow hMSCs. Compared with nonfailing hMSCs, failing hMSCs prolonged action potential duration by 24% (P<0.001, n=15), increased Ca2+ alternans by 300% (P<0.001, n=18), and promoted spontaneous calcium release activity (n=14, P<0.013) in human cardiac myocytes derived from induced pluripotent stem cells. Failing hMSCs exhibited increased secretion of inflammatory cytokines IL (interleukin)-1β (98%, P<0.0001) and IL-6 (460%, P<0.02) compared with nonfailing hMSCs. IL-1β or IL-6 in the absence of hMSCs prolonged action potential duration but only IL-6 increased Ca2+ alternans and promoted spontaneous calcium release activity in human cardiac myocytes derived from induced pluripotent stem cells, replicating the effects of failing hMSCs. In contrast, nonfailing hMSCs prevented Ca2+ alternans in human cardiac myocytes derived from induced pluripotent stem cells during oxidative stress. Finally, nonfailing hMSCs exhibited >25× higher secretion of IGF (insulin-like growth factor)-1 compared with failing hMSCs. Importantly, IGF-1 supplementation or anti-IL-6 treatment rescued the arrhythmia substrates induced by failing hMSCs. We identified device leads as a novel source of cardiac hMSCs. Our findings show that cardiac hMSCs can regulate arrhythmia substrates by remodeling their secretome in disease. Importantly, therapy inhibiting (anti-IL-6) or mimicking (IGF-1) the cardiac hMSC secretome can rescue arrhythmia substrates.

Glutamine Depletion By Addicted Myeloma Cells Inhibits Osteoblastic Differentiation of Bone Marrow Mesenchymal Stromal Cells Limiting Asparagine Availability: A Possible New Mechanism for Myeloma Bone Disease

Metabolic alterations of cancer cells, aimed at sustaining their growth, may also influence the behavior of the tumor microenvironment. Our group has recently demonstrated that multiple myeloma (MM) is a highly glutamine(Gln)-addicted tumor that utilize huge amounts of Gln to fuel its metabolism through the enzyme glutaminase (GLS). For this reason, MM cells exhibits increased Gln uptake, mainly through the ASCT2 transporter. Interestingly, lower bone marrow (BM) plasma Gln concentration (down to a median value of 0.4 mM vs. a median value of 0.6 mM) was found in MM patients as compared with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS). The main feature of MM BM microenvironment is the suppression of osteoblastic (OB) differentiation leading to the development of osteolytic bone lesions, the hallmark of MM. Most recently, it has been demonstrated that Gln metabolism is needed to sustain bone mass formation in murine models and that GLS inhibition decreases OB differentiation of human mesenchymal stromal cells (hMSCs). However, no information is yet available on the role of Gln depletion imposed by MM cell metabolism on OB differentiation into the BM. This topic has been investigated in the present study. Firstly, human MM cells were co-cultured with BM hMSCs, and Gln medium concentration was evaluated with mass spectrometry (MS), demonstrating a MM-induced depletion of the amino acid. Upon Gln depletion, MSC exhibited a sustained induction of Glutamine Synthetase (GS). On the contrary, when differentiated in osteogenic medium (D-MEM + 5% Fetal Bovine Serum, supplemented with 2 mM Gln, ascorbic acid and dexamethasone), GS was suppressed. Conversely, GLS (both KGA and GAC isoforms) and SLC38A2, the gene for the concentrative Gln transporter SNAT2, were induced. These data suggest that hMSCs differentiation in OBs is associated with an increased dependence upon extracellular Gln. Consistent with this conclusion, the activity of SNAT2 was absent in undifferentiated hMSCs but well detectable after 14 days of OB differentiation, when total Gln uptake was also increased. Under the same conditions, OB differentiation markers (RUNX2, COL1A1, ALPL expression and ALPL activity or staining) were significantly induced but their expression was blunted by incubation in low-Gln (0.4 mM) medium or in the presence of the SNAT2 inhibitor MeAIB. The incubation in Gln-free D-MEM suppressed the induction of GLS and SLC38A2 along with OB differentiation, which was restored by the supplementation of Non-Essential Amino Acids (NEAA). Among NEAA, only asparagine (Asn) was able to rescue OB differentiation in the absence of Gln. The determination of intracellular amino acids with MS indicated that OB differentiation was associated with the increase of cell Asn, without significant changes of Gln, glutamate (Glu) or aspartate (Asp). Asparagine Synthetase (ASNS), the Gln-dependent enzyme that accounts for Asn synthesis, was also found induced during OB differentiation of hMSCs. Gene Expression Profiles of primary BM hMSCs and OBs from bone biopsies of both healthy donors (n=7) and MM patients (n=16) indicated that GLS, ASNS, and SLC38A2 are more expressed in OBs, while the expression of GLUL, the gene for GS, is higher in undifferentiated hMSCs from healthy donors. Overall, these results indicate that (1) OB differentiation of hMSCs is Gln-dependent; (2) the partial Gln depletion, imposed by Gln-addicted MM cells in the BM microenvironment, contributes to the impairment of osteoblastic differentiation of hMSCs; (3) hindrance of differentiation may depend on the limited availability of intracellular Asn derived from Gln-dependent ASNS. These results support the evidence that Gln addiction of MM cells affects bone microenvironment leading to the inhibition of OB differentiation and, consequently, to the development of MM bone disease. Disclosures Giuliani: Janssen: Research Funding.

Human mesenchymal stem cells maintain their phenotype, multipotentiality, and genetic stability when cultured using a defined xeno-free human plasma fraction

BackgroundMesenchymal stem cells (MSCs) show promising characteristics for their use in advanced therapy medicinal products. However, there are some unresolved concerns, such as the use of animal components for their expansion.In this study we assessed the suitability of a xeno-free supplement for cell culture (SCC) derived from human plasma, to culture and expand human MSCs (hMSCs) from different origins. Characteristics of viable cultured hMSCs such as genetic stability, phenotype and multipotentiality were qualitatively evaluated.MethodshMSCs from adipose tissue (AT), bone marrow (BM) and umbilical cord (UC) and supplier sources (commercial/non-commercial) were used. After hMSCs expansion in a xeno-free medium, classical hMSCs markers were studied by immunocytochemistry, and genetic stability was tested by classic karyotyping. The capacity of hMSCs to differentiate into adipogenic, osteogenic, and chondrogenic cells in differentiation media was assessed using different staining. Different lots of SCC were used to assure consistency between batches.ResultsAll hMSCs tested maintained their morphology and adherence to plastic during their expansion, and preserved their genetic stability, phenotype and differentiation potential. No differences were observed when using different lots of SCC. Moreover, the proliferation rate, evaluated as population doubling time (PDT) of commercial BM and AT hMSCs, was higher in the xeno-free medium than in the control media provided by the suppliers of the cells (PDT of 4.6 for BM-hMSC and 6.4 for AT-hMSC in xeno-free medium, and 7.0 and 14.7 respectively in the commercial media). UC-hMSCs PDT was similar in all the media tested. When using non-commercial BM-hMSCs, PDT was lower in the xeno-free medium, but reverted to the control level with the addition of growth factors.ConclusionsSCC-containing medium can be a feasible xeno-free alternative to expand hMSCs for advanced therapies.

Involvement of Notch signaling in the osteogenic differentiation of human mesenchymal stem cells stimulated by pulsed electromagnetic fields

Biophysical stimulation with pulsed electromagnetic fields (PEMFs), used in clinics to promote bone repair, favour osteogenic differentiation in human mesenchymal stem cells (hMSCs), however their molecular mechanisms are not clarified. Notch is a pathway regulating cell fate decisions which play a role in skeletal development. Notch signaling is initiated by binding a Notch ligand to a cell surface Notch receptor, resulting in a cleavage of receptor and releasing Notch intracellular domain which translocates to the nucleus and activates transcription of nuclear Notch target genes, such as the Hes/Hey family.The aim of this study is to establish if the known PEMF-induced osteogenic effects may occur through the modulation of Notch pathway. Bone marrow hMSCs cultured in basal condition (control) and in osteoinductive medium (OM) for 28 days were unexposed or continuously exposed to PEMFs (75 Hz, 1.5 mT) (Igea, Carpi, Italy). To block Notch pathway, the Notch inhibitor DAPT was used to treat a series of hMSCs cultured in OM. At different time points (day 1,3,7,14,21,28), osteogenic markers (alkaline phosphatase activity, osteocalcin and matrix mineralization), mRNA expression of osteogenic transcription factors (Runx2, Dlx5, Osterix) as well as of Notch receptors (Notch1-4), their ligands (Jagged1, Dll1 and Dll4) and nuclear target genes (Hey1, Hey2, Hes1, Hes5) were analysed.Our results showed that osteogenic markers and transcription factors increased in OM compared to control and they were further stimulated by PEMFs. Notably, PEMFs significantly increased the expression of Notch4, Dll4, Hey1, Hes1 and Hes5 in the middle phase of differentiation in OM compared to control. In the presence of DAPT, osteogenic markers as well Hes1 and Hes5 expression were significantly inhibited, in unexposed and PEMF-exposed hMSCs. Hey1 was not inhibited by DAPT suggesting a possible regulation by other signaling pathway.These new findings show that PEMFs favor osteogenic differentiation acting through Notch pathway, adding important knowledge concerning the molecular mechanisms by which PEMFs can modulate osteogenesis. This work was supported by grants from IGEA, Carpi, Italy.

Effect of gold nanoparticles on adipogenic differentiation of human mesenchymal stem cells

Gold nanoparticles are very attractive for biomedical products. However, there is a serious lack of information concerning the biological activity of nanosized gold in human tissue cells. An influence of nanoparticles on stem cells might lead to unforeseen consequences to organ and tissue functions as long as all cells arising from the initial stem cell might be subsequently damaged. Therefore the effect of negatively charged gold nanoparticles (9 and 95 nm), which are certified as reference material for preclinical biomedical research, on the adipogenic differentiation of human mesenchymal stem cells (hMSCs) is investigated here. Bone marrow hMSCs are chosen as differentiation model since bone marrow hMSCs are well characterized and their differentiation into the adipogenic lineage shows clear and easily detectable differentiation. In this study effects of gold nanoparticles on adipogenic differentiation are analyzed regarding fat storage and mitochondrial activity after different exposure times (4–21 days). Using time lapse microscopy the differentiation progress under chronically gold nanoparticle treatment is continuously investigated. In this preliminary study, chronically treatment of adipogenic differentiating hMSCs with gold nanoparticles resulted in a reduced number and size of lipid vacuoles and reduced mitochondrial activity depending on the applied concentration and the surface charge of the particles.

Chondrogenesis and Mineralization DuringIn VitroCulture of Human Mesenchymal Stem Cells on Three-Dimensional Woven Scaffolds

Human mesenchymal stem cells (hMSCs) and three-dimensional (3D) woven poly(ɛ-caprolactone) (PCL) scaffolds are promising tools for skeletal tissue engineering. We hypothesized that in vitro culture duration and medium additives can individually and interactively influence the structure, composition, mechanical, and molecular properties of engineered tissues based on hMSCs and 3D poly(ɛ-caprolactone). Bone marrow hMSCs were suspended in collagen gel, seeded on scaffolds, and cultured for 1, 21, or 45 days under chondrogenic and/or osteogenic conditions. Structure, composition, biomechanics, and gene expression were analyzed. In chondrogenic medium, cartilaginous tissue formed by day 21, and hypertrophic mineralization was observed in the newly formed extracellular matrix at the interface with underlying scaffold by day 45. Glycosaminoglycan, hydroxyproline, and calcium contents, and alkaline phosphatase activity depended on culture duration and medium additives, with significant interactive effects (all p < 0.0001). The 45-day constructs exhibited mechanical properties on the order of magnitude of native articular cartilage (aggregate, Young's, and shear moduli of 0.15, 0.12, and 0.033 MPa, respectively). Gene expression was characteristic of chondrogenesis and endochondral bone formation, with sequential regulation of Sox-9, collagen type II, aggrecan, core binding factor alpha 1 (Cbfα1)/Runx2, bone sialoprotein, bone morphogenetic protein-2, and osteocalcin. In contrast, osteogenic medium produced limited osteogenesis. Long-term culture of hMSC on 3D scaffolds resulted in chondrogenesis and regional mineralization at the interface between soft, newly formed engineered cartilage, and stiffer underlying scaffold. These findings merit consideration when developing grafts for osteochondral defect repair.

The In Vitro Migration Capacity of Human Bone Marrow-Derived Mesenchymal Stem Cells in Response to Chemokines and Mesenchymal Growth Factors.

Bone marrow (BM) mesenchymal stem cells (MSCs) are characterized by their wide differentiation potential into adipose, bone, cartilage, muscle, and neural tissues. This capacity makes MSCs potentially useful for cell therapy in case of tissue repair. The therapeutic efficacy of such cells depends on their capacity to migrate to damaged tissues in response to pro-migratory signals produced locally. In this study, we investigated the in vitro migration capacity of human BM MSCs (hMSCs) evaluating their response to several potential chemotactic factors, including chemokines and mesenchymal growth factors (MGF). BM cells obtained from patients undergoing orthopedic surgery, were cultured in alpha-MEM medium with 10% fetal calf serum (FCS). Non adherent cells were discarded after 2–3 days. Layers were passaged once or twice to deplete in hematopoietic cells. The three-lineal (adipogenic, osteoblastic, and chondrocytic) differentiation potential of CD45- cells was verified. hMSCs were then induced to migrate (37°C, 5% CO2 for 16 hours) through Transwells (8-μm pore filters), in response to purified chemotactic agents at optimal concentrations, as compared to that of medium with 30% FCS (positive control) and to that of medium alone (negative control). In parallel, the pattern of chemokine and growth factor receptor expression was assessed by flow cytometry and transcripts for cytokines, chemokines and receptors were quantified using Taqman Low Density Arrays® (RQ-PCR). Among MGF, the best pro-migratory activity was observed with PDGF-AB and IGF-1 (mean values of 64.9 and 37.9% of the positive control, respectively), while EGF and HGF activity was 20%–30% of the positive control and VEGF and FGF2 activity was comparable to that of the negative control (10%). Among chemokines, only MCP-1, Eotaxin-2 and MDC had significant activity (about 20 % of the control), while activity of RANTES and Eotaxin-1 was between 10 and 20% and that of SDF-1, Fractalkine, MIP-1alpha and GROalpha was below 10%. CCR1, CCR3, CCR4, CXCR4, PDGF-R, HGF-R, EGF-R and IGF1-R were expressed by hMSCs, as demonstrated by flow cytometry. RQ-PCR analysis indicated that some chemokines were expressed at high level (X-Y level of the GAPDH housekeeping gene), either constitutively (MCP-1, GROalpha), or after IL-1/TNF-alpha stimulation (RANTES, SDF-1), while others were expressed at intermediate (Fractalkine) or low (MIP-1alpha) level. Chemokine receptors were usually expressed at very low level (X-Y level of GAPDH), either constitutively (CCR3, CX3CR1) or only after stimulation (CCR1, CCR4, CXCR4 and CXCR5), while CCR2, CXCR-1 and CXCR-2 were not expressed at all. Transcripts for all MGF assayed in vitro and for their receptors were detected at intermediate/high level. In conclusion, mesenchymal growth factors appear to be better chemoattractants in vitro for BM hMSCs than chemokines. The reduced pro-migratory activity of exogenous chemokines might be related to the low level of their receptors, themselves downregulated by endogenous chemokines autocrinally produced.