Marrow-derived Mesenchymal Stromal Cells Research Articles

B-cell precursor acute lymphoblastic leukemia elicits an interferon-α/β response in bone marrow-derived mesenchymal stroma.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) can hijack the normal bone marrow microenvironment to create a leukemic niche which facilitates blast cell survival and promotes drug resistance. Bone marrow-derived mesenchymal stromal cells (MSCs) mimic this protective environment in ex vivo co-cultures with leukemic cells obtained from children with newly diagnosed BCP-ALL. We examined the potential mechanisms of this protection by RNA sequencing of flowsorted MSCs after co-culture with BCP-ALL cells. Leukemic cells induced an interferon (IFN)-related gene signature in MSCs, which was partially dependent on direct cell-cell signaling. The signature was selectively induced by BCP-ALL cells, most profoundly by ETV6-RUNX1 positive ALL cells, as coculture of MSCs with healthy immune cells did not provoke a similar IFN signature. Leukemic cells and MSCs both secreted IFNα and IFNβ, but no IFNγ. In line, the IFN-gene signature was sensitive to blockade of IFNα/β signaling, but less to that of IFNγ. The viability of leukemic cells and level of resistance to three chemotherapeutic agents was not affected by interference with IFN signaling using selective IFNα/β inhibitors or silencing of IFN-related genes. Taken together, our data suggest that the leukemia-induced expression of IFNα/β-related genes by MSCs does not support survival of BCPALL cells but may serve a different role in the pathobiology of BCP-ALL.

Genetic Subtype Driven Changes in Bone Marrow Stromal Cells of Children with B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction We previously showed that B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells hijack the normal bone marrow microenvironment to create a leukemic niche which facilitates blast cell survival and promotes drug resistance [1-3]. Bone marrow-derived mesenchymal stromal cells (MSCs) are a major component of the bone marrow microenvironment . Ex vivo,these cells provide a survival benefit to co-cultured leukemic cells and induce resistance to chemotherapeutic drugs [1, 2]. We previously showed that leukemic cells re-sensitize to chemotherapeutics ex vivo when the interaction with MSCs is disrupted [2]. Results We here report that patients' BCP-ALL cells induce an Interferon (IFN)-related gene signature in patients'-derived MSCs; a.o. IFI6, MX1, and OAS3 were 4.3 to 7.7-fold upregulated in MSCs flow-sorted after co-culture with BCP-ALL cells compared to the same MSCs flow-sorted after mono-culture . BCP-ALL samples (n=15) with an ETV6-RUNX1 translocation (5 out of 8 samples) were the most potent inducers of the IFN-related gene expression signature in MSCs compared to B-other BCP-ALL (1 out of 6 samples), with IFI6 3.0-fold (p=0.03), MX1 2.6-fold(p=0.05) and OAS3 2.1-fold (p=0.04) higher in MSCs after co-culture with ETV6-RUNX1 BCP-ALL cells. Similarly, an ETV6-RUNX1cell line (REH) also induced this IFN-response more strongly in MSCs compared to cell lines representing other BCP-ALL subtypes (SUPB15, MUTZ5, RCH-ACV and NALM6). Addition of specific inhibitors to these ex vivo BCP-ALL and MSCs cultures, revealed that the ETV6-RUNX1 induced IFN signature was dependent on IFNα/β signaling, whereas no such effect was seen for inhibitors of IFNγ signaling. Additionally, inhibition of direct contact between BCP-ALL cells and MSCs also negatively affected the expression of some IFN responsive genes in MSCs ( CXCL10 3.3-fold, p=0.02, IFI44L 1.57-fold, p=0.01 and IFITM1 1.5-fold, p=0.005) while other genes remained unaltered, suggesting that the transcriptional changes in MSCs are driven by close contact with BCP-ALL cells and not by soluble factors released by these cells or those being part of the culture medium. Normal immune cells including various populations of T-cells, monocytes, NK cells and dendritic cells did not alter the expression levels of the IFN-related genes in MSCs. We also observed that, while MSCs provide a survival benefit to (ETV6-RUNX1) BCP-ALL cells and induce resistance to chemotherapeutic drugs (prednisolone, daunorubicin, L-asparaginase) in these leukemic cells, this was not counteracted by inhibition of IFNα/β signaling. Conclusion Our data show that leukemic cells, more specifically ETV6-RUNX1 positive BCP-ALL cells, induce an IFNα/β signature in MSCs. Activation of this IFNα/β pathway in MSCs does not cause resistance of BCP-ALL cells to chemotherapeutic drugs and hence the IFNα/β activation may serve a different role, e.g., affecting the migration (recruitment and/or repelling) of specific immune cells to the leukemic bone marrow niche. Our findings warrant further studies into the role of the BCP-ALL-induced IFNα/β response in chemotaxis of healthy immune cells to create a beneficial microenvironment for BCP-ALL cells. This knowledge might point to alternative, maybe less intensive ways to eradicate leukemic cells.

RUNX1::RUNX1T1 Impairs the Differentiation Potential of Primary AML Cells

Acute myeloid leukemias (AMLs) are often driven by genomic translocations, of which t(8;21) is the most frequent. The resulting gene fusion RUNX1::RUNX1T has been shown in cell line and transgenic expression models to impede cell differentiation and promote leukemic self-renewal. However, it has remained unclear if and how RUNX1::RUNX1T1 influences leukemic propagation in patient-derived AML cells. To address the role of RUNX1::RUNX1T1 in the differentiation status and heterogeneity of patient-derived leukemic blasts, we developed an siRNA lipid nanoparticle (LNP) delivery system for the fusion transcript knockdown (Issa et al, Leukemia 2023). For efficient delivery to primary cells, the LNPs were functionalized with a Leu-Asp-Val (LDV) tripeptide binding the Very Late antigen-4 receptor on leukocytes. Primary AML cells were cultured and expanded for several weeks using a serum-free co-culture system with human bone marrow-derived mesenchymal stromal cells (MSCs) (Swart et al, Pharmaceutics 2023). Treatment of t(8;21) patient-derived xenograft (PDX) and primary bone marrow aspirates depleted RUNX1::RUNX1T1 transcript and protein, as determined by RT-qPCR and western blotting. We unraveled the transcriptional repercussions of RUNX1::RUNX1T1 knockdown using bulk RNAseq and ATACseq in a PDX and identified 1068 up- and 1521 downregulated genes upon RUNX1::RUNX1T1 knockdown, as compared to mismatched siRNA-treated cells (padj < 0.001, log2FC > 2). Gene set enrichment analysis revealed loss of self-renewal potential and enhanced myeloid differentiation. Similarly, ATACseq revealed an increased GMP/monocyte-like chromatin accessibility and a decreased HCS/MPP-like one upon RUNX1::RUNX1T1 knockdown (Figure 1). Surprisingly and in contrast to previous cell line results, RUNX1::RUNX1T1 depletion correlated with increased cell cycle and DNA repair signatures. These combined data demonstrate that RUNX1::RUNX1T1 perturbance promotes differentiation of leukemic stem cells towards granulocytic and monocytic lineages. Furthermore, they suggest that the initial stages of differentiation are linked to increased proliferation in line with the increased proliferation capacity of GMPs compared to more immature progenitor states. To investigate the impact of RUNX1::RUNX1T1 on leukemic blast populations at greater granularity, we knocked RUNX1::RUNX1T1 down in 2 primary bone marrow aspirates and 1 PDX and performed single-cell (sc) RNAseq. Notably, principal component analysis revealed that loss of RUNX1::RUNX1T1 shifted all leukemic populations, while hardly affecting normal hematopoietic cells or MSCs (Figure 2). In all samples, pseudotime analysis showed a more differentiated state of cells upon RUNX1::RUNX1T1 depletion. For cell type annotation, we projected our datasets onto a reference normal bone marrow dataset (van Galen et al, Cell 2019). This projection confirmed differentiation toward granulocyte-monocyte progenitors (GMPs), granulocytes and monocytes. Strikingly, the confidence of cell type prediction according to the reference normal bone marrow was significantly and substantially higher upon RUNX1::RUNX1T1 depletion than in cells with unimpaired fusion transcript level, suggesting restoration of normal myeloid differentiation. In addition to that, primary RUNX1::RUNX1T1 AML samples also contained a new, distinct cell subpopulation that emerged upon RUNX1::RUNX1T1 depletion. That subpopulation could not be annotated as resembling any normal bone marrow cell type. However, it expressed eosinophil marker genes such as RNASE3 and EPX. We, therefore, labeled this cell subpopulation as aberrant eosinophilic. This reveals RUNX1::RUNX1T1's role in disrupting differentiation towards multiple lineages. Our work illustrates a strong dependency of the differentiation potential of t(8;21) AML cells on their driver gene RUNX1::RUNX1T1 in an ex vivo setting with PDX or primary cells. We demonstrate that RUNX1::RUNX1T1 hijacks more than one differentiation trajectory of the AML blasts. Our findings also highlight siRNA-loaded LNPs as an innovative therapeutic approach for fusion gene-driven AML by directly targeting the fusion gene as the leukemic driver.

Pre-cultured, cell-encapsulating fibrin microbeads for the vascularization of ischemic tissues.

There is a significant clinical need to develop effective vascularization strategies for tissue engineering and the treatment of ischemic pathologies. In patients afflicted with critical limb ischemia, comorbidities may limit common revascularization strategies. Cell-encapsulating modular microbeads possess a variety of advantageous properties, including the ability to support prevascularization in vitro while retaining the ability to be injected in a minimally invasive manner in vivo. Here, fibrin microbeads containing human umbilical vein endothelial cells (HUVEC) and bone marrow-derived mesenchymal stromal cells (MSC) were cultured in suspension for 3 days (D3 PC microbeads) before being implanted within intramuscular pockets in a SCID mouse model of hindlimb ischemia. By 14 days post-surgery, animals treated with D3 PC microbeads showed increased macroscopic reperfusion of ischemic foot pads and improved limb salvage compared to the cellular controls. Delivery of HUVEC and MSC via microbeads led to the formation of extensive microvascular networks throughout the implants. Engineered vessels of human origins showed evidence of inosculation with host vasculature, as indicated by erythrocytes present in hCD31+ vessels. Over time, the total number of human-derived vessels within the implant region decreased as networks remodeled and an increase in mature, pericyte-supported vascular structures was observed. Our findings highlight the potential therapeutic benefit of developing modular, prevascularized microbeads as a minimally invasive therapeutic for treating ischemic tissues.

Clinical use of autologous adipose-derived stromal vascular fraction cell injections for hip osteoarthritis

IntroductionCurrently, studies on adipose-derived stromal vascular fraction (SVF) cells are attracting increasing attention because they have the potential to differentiate into a subset of cell types, such as bone marrow-derived mesenchymal stromal cells (MSCs), and are easier to harvest than MSCs, thus making them easier to apply clinically. This study evaluated the short-term clinical outcomes of SVF cell therapy for hip osteoarthritis (OA). MethodsForty-two patients were enrolled in this study; these patients received a single injection comprising an average of 3.8 (standard deviation [SD], ±1.3) × 107 SVF cells into the hip joint. All patients were followed-up for at least 6 months. The mean age of the patients was 60.2 years (SD, ±9.4 years). Kellgren–Lawrence (KL) grades II, III, and IV based on radiography were 13, 13, and 16 patients, respectively. SVF cells were obtained from the subcutaneous fat of the abdomen or breech using a Celution® 800/CRS system. The average cell viability of SVF cells was 90.8% (SD, ±2.8%). Clinical assessments were performed using the Harris Hip Score (HHS), Japanese Orthopaedic Association Hip Disease Evaluation Questionnaire (JHEQ) score, and visual analog scale (VAS) score to evaluate pain. Images were evaluated using radiography, and T2 mapping values were obtained using a 1.5-T magnetic resonance imaging system. These clinical and imaging assessments were followed from preoperatively to 6 months postoperatively. ResultsThe HHS, JHEQ score, and VAS score improved significantly from 22.5 (SD, ±16.6), 26.6 (SD, ±11.3), and 75.5 (SD, ±15.8) preoperatively to 46.8 (SD, ±27.2), 39.4 (SD, ±19.7), and 46.5 (SD, ±27.9), respectively, at 6 months postoperatively. KL grade II showed significant improvement in clinical outcome from preoperative to postoperative, while KL grade IV showed slight or little improvement. The center edge angle, acetabular head index on the radiographs, and T2 mapping values did not change significantly from preoperatively to 6 months postoperatively. ConclusionsSVF cell injection in the hip joint showed good short-term clinical efficacy for reducing hip OA symptoms. SVF cell therapy is thus an innovative and effective treatment for hip OA.

Comparison between articular chondrocytes and mesenchymal stromal cells for the production of articular cartilage implants.

Focal lesions of articular cartilage give rise to pain and reduced joint function and may, if left untreated, lead to osteoarthritis. Implantation of in vitro generated, scaffold-free autologous cartilage discs may represent the best treatment option. Here we compare articular chondrocytes (ACs) and bone marrow-derived mesenchymal stromal cells (MSCs) for their ability to make scaffold-free cartilage discs. Articular chondrocytes produced more extracellular matrix per seeded cell than mesenchymal stromal cells. Quantitative proteomics analysis showed that articular chondrocyte discs contained more articular cartilage proteins, while mesenchymal stromal cell discs had more proteins associated with cartilage hypertrophy and bone formation. Sequencing analysis revealed more microRNAs associated with normal cartilage in articular chondrocyte discs, and large-scale target predictions, performed for the first time for in vitro chondrogenesis, suggested that differential expression of microRNAs in the two disc types were important mechanisms behind differential synthesis of proteins. We conclude that articular chondrocytes should be preferred over mesenchymal stromal cells for tissue engineering of articular cartilage.

Comparative in vitro treatment of mesenchymal stromal cells with GDF-5 and R57A induces chondrogenic differentiation while limiting chondrogenic hypertrophy

PurposeHypertrophic cartilage is an important characteristic of osteoarthritis and can often be found in patients suffering from osteoarthritis. Although the exact pathomechanism remains poorly understood, hypertrophic de-differentiation of chondrocytes also poses a major challenge in the cell-based repair of hyaline cartilage using mesenchymal stromal cells (MSCs). While different members of the transforming growth factor beta (TGF-β) family have been shown to promote chondrogenesis in MSCs, the transition into a hypertrophic phenotype remains a problem. To further examine this topic we compared the effects of the transcription growth and differentiation factor 5 (GDF-5) and the mutant R57A on in vitro chondrogenesis in MSCs.MethodsBone marrow-derived MSCs (BMSCs) were placed in pellet culture and in-cubated in chondrogenic differentiation medium containing R57A, GDF-5 and TGF-ß1 for 21 days. Chondrogenesis was examined histologically, immunohistochemically, through biochemical assays and by RT-qPCR regarding the expression of chondrogenic marker genes.ResultsTreatment of BMSCs with R57A led to a dose dependent induction of chondrogenesis in BMSCs. Biochemical assays also showed an elevated glycosaminoglycan (GAG) content and expression of chondrogenic marker genes in corresponding pellets. While treatment with R57A led to superior chondrogenic differentiation compared to treatment with the GDF-5 wild type and similar levels compared to incubation with TGF-ß1, levels of chondrogenic hypertrophy were lower after induction with R57A and the GDF-5 wild type.ConclusionsR57A is a stronger inducer of chondrogenesis in BMSCs than the GDF-5 wild type while leading to lower levels of chondrogenic hypertrophy in comparison with TGF-ß1.

Mesenchymal stem cell therapy in patients with Crohn’s disease — 5 year follow-up

Abstract We investigated the efficacy of combination therapy using bone marrow-derived mesenchymal stromal cells (MSC) and Infliximab (IFX) to achieve “deep remission” in patients with luminal Crohn disease (CD). Our study included 72 patients (age 19–62 years) (Ме = 29) with luminal CD. Patients in the 1st group (n = 21) received standard 5-aminosalicylic acid (5-ASA) and glucocorticosteroid therapy in combination with MSC. Patients in the 2nd group (n = 32) were prescribed anti-cytokine therapy IFX. Patients in the 3rd group (n = 19) received MSC and IFX. Clinical and immunobiological (C-reactive protein-CRP and faecal calprotectin-FCP) showed a more significant decrease of local and systemic inflammation activity in the 3rd group of patients. During a 5-year follow-up we observed longer duration of remission in patients who received MSC and IFX compared to the 1st (р = 0,04) and 2nd groups of patients (р = 0.038). Combination therapy of bone marrow-derived MSC and IFL provides “deep remission” in patients with luminal CD and has higher prognostic value for duration of CD remission period.

Inhibition of the Menin-MLL1 Interaction in MLL-Rearranged Primary Mixed-Phenotype Acute Leukemia Samples Promotes Leukemic Differentiation

Mixed phenotype acute leukemias (MPALs) simultaneously present features typical for more than one hematological cell lineage. The rarity of such patients combined with the lack of well-defined diagnostic criteria have so far prevented the design of optimal therapy regimens resulting in the occurrence of lineage switch and therewith associated treatment failures. Rearrangements involving MLL gene (also known as KMT2A) occur in around 15% of MPAL patients (typically infants) and are associated with a very poor outcome. Moreover, several studies demonstrated dependency of MLL-rearranged (MLL-r) leukaemias on the interaction between N-terminus of MLL with Menin and its successful therapeutic targeting by small molecular weight compounds, that are currently validated in clinical trials. Here we investigated potential application of the Menin-MLL inhibitor SNDX-5613 in MLL/AF4 MPAL infants who presented with the bilineage B/Myeloid phenotype. Using a co-culture approach with human bone marrow-derived mesenchymal stromal cells (MSCs) and media optimized for growth of lymphoid and myeloid lineages, we were able to expand and monitor primary MLL-r patient material for more than three weeks. Primary material from two MPAL patients and their lymphoid and myeloid subpopulations were analyzed for their drug responses including immunophenotypic and global transcriptomic characterization. We observed a dramatic loss of cell numbers in response to SNDX-5613, with the IC50 value ranging from 80 to 800nM. Similar analyses of the sorted fractions revealed that the observed loss of cell numbers is mainly caused by the reduction of the lymphoid subfraction. Immunophenotypic analyses showed that surviving cells within both lymphoid and myeloid fractions are losing CD34 with simultaneous gain of the CD38 marker, suggesting cellular maturation. Treatment of the lymphoid fraction with SNDX-5613 caused a dose-dependent loss of the CD19+CD33+ cell population and a simultaneous gain of CD19+CD33-, and CD19-CD33+ cells. These data suggest the cellular origin of the myeloid fraction to be residing within the lymphoid counterpart. Interestingly, myeloid cells responded to Menin-MLL inhibition with increased surface expression of monocytic markers (e.g. CD11b, CD14, CD15), elevated RNA expression of genes involved in cell adhesion and morphological changes, indicating myeloid differentiation of the surviving cells. Moreover, transcriptomic analysis of the lymphoid subfraction revealed dose-dependent increase of the MS4A1 gene expression (encoding for CD20) suggesting simultaneous lymphoid maturation of the CD19+ proB-like blasts. In conclusion, our study shows that Menin-MLL inhibition drives both myeloid differentiation and lymphoid maturation in MLL-r MPALs. These myeloid data are consistent with observations of differentiation syndrome as an on-target effect in current clinical trials while the phenotypic changes in the lymphoid cells are new findings. Together, these data strongly suggests that MLL-r MPAL patients could benefit from the inclusion of the Menin-MLL1 inhibitors into their treatment regimens.

Novel Drugs in the Treatment of Steroid Refractory Acute Graft Versus Host Disease

Steroid refractory acute graft versus host disease (SR-aGVHD) remains an area of unmet needs with more therapeutic options required. Here, we review novel treatment options for SR-aGVHD. Methods: A comprehensive data search was done across various data sets, including PubMed, Cochrane, and Embase, using MeSH terms and keywords to look for all the different drug types that have been studied for SR-aGVHD. A summary of the results is described here. Results: Denilekin diftitox (Ontak), a recombinant protein composed of IL-2 fused to diphtheria toxin, was studied in a phase 1 trial by Vincent et al. Thirty patients receiving 9 g/kg of Ontak at different intervals showed a Complete response (CR) of 38% and a partial response (PR) of 38%. Remetemcel-L, ex vivo cultured adult human mesenchymal stem cells were studied in a multicenter phase III trial in 260 patients by Kebraei et al. compared to placebo. At a dose of 2 x 106 hMSC/kg, an overall response rate (ORR) of 58% vs. 54% in the placebo group was seen with CR of 35 and 30% respectively at day 28. Third-party bone marrow-derived mesenchymal stromal cells (MSC) expanded in platelet lysate were studied by Introna et al. in a phase 1 trial. Forty patients received a median dose of 1.5 x 10(6)/kg per infusion and showed an ORR of 67.5% with a CR of 27.5%. OS at 1 and 2 years was 50% and 38.6%, respectively. Pentostatin, an adenosine deaminase inhibitor, was studied by Meade et al. in a phase 1 trial in 23 patients. Pentostatin was administered at a dose of 1 to 4 mg/m2/day. Study showed a CR of 63%. Sirolimus, an mTOR Inhibitor, was studied by Benito et al. in a pilot study in 21 patients. On an average dose of 5 mg/m2, sirolimus showed a CR of 23% and PR of 33%. Couriel et al., in a phase III trial, 63 patients were randomized and received either methylprednisone (MP) 2mg/kg or infliximab plus MP. On days 7 and 28 response rate for infliximab + MP vs. MP was 52% vs. 78% and 62% vs. 58%, respectively. Przepiorka et al. reported the results of an open-label phase II study in 20 patients with SR-aGVHD treated with BTI-322 (LO-CD2a), an anti-human CD2 SR-aGVHD IgG2b monoclonal antibody (MAB). ORR was achieved in 55% of the patients. Deeg et al. resulted from a dose-escalation study in 59 patients with steroid-refractory aGVHD treated with ABX-CBL MAB. Reported ORR was 51%. Carpent et al. reported the results of a phase II trial in which 44 patients were treated with visilizumab with ORR of 32% at day 42. Bordigoni et al. treated 62 patients with daclizumab in a phase II trial with reportef ORR of 68.80% at 44 months follow up. Groth et al. studied anti-CD3, anti CD7 antibodies separately conjugated to recombinant ricin A (CD3/CD7-1 T) in adults, 18 years and older with grade III to IV aGVHD following HSCT for myeloid and lymphoid malignancies. Twenty patients were treated with an ORR of 60% on day 28. Adverse events are listed in the Table 2. Conclusion Different targeted drugs focused on the specific pathways show promising results. Monoclonal antibodies in particular showed good treatment outcomes with tolerable safety profile. Further studies are required to identify a valuable therapeutic option, SR-aGVHD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Investigating the Relationship between Primary Bone Marrow Adipocytes and Myeloma Cells with Three Cell Culture Systems

Introduction & Aims of the Study: The unique properties of the bone marrow allow for migration and proliferation of multiple myeloma (MM) cells, while also providing the ideal environment for development of quiescent, drug-resistant MM cell clones. Recent studies suggest that bone marrow adipocytes (BMAds) can contribute to myeloma-induced bone disease, and reciprocally, that BMAds support MM cells in vitro. Importantly, most data investigating BMAds have been generated using adipocytes differentiated from bone marrow-derived mesenchymal stromal cells (MSCs) in vitro due to the technical challenges associated with isolating and culturing primary adipocytes. To that end, we investigated the MM-BMAd relationship ex vivo utilizing primary adipocytes (pBMAds), which overcome many of the drawbacks associated with using MSC-derived adipocytes. Methods: pBMAds were isolated from the top of human bone marrow biopsies after centrifugation and seeded onto one of three in vitro systems. To develop an in vitro model with a tissue-like structure to mimic the bone marrow microenvironment, we developed the first 3D, tissue engineered model utilizing pBMAds derived from human bone marrow. Following adherence of pBMAds, MM cells were added to investigate bidirectional tumor-host signaling. Viable pBMAds were measured utilizing CellTiterGlo and RealTime Glo (bioluminescence), alone and in culture with MM cells (MM.1Sgfp+luc+ and RPMI-8226). MM.1Sgfp+luc+ cell number was determined by luciferin spike-in and measurement of bioluminescence. To investigate the effects of myeloma cells on pBMAds, we used fluorescence and confocal microscopy to image cell morphology, and mass spectrometry proteomics of conditioned media samples to specifically interrogate soluble signals. Gene expression in both cell types was investigated by qPCR. Results: Utilizing adipocytes isolated from 12 different human donors, we found that pBMAds, which are extremely fragile, can be isolated and stably cultured in 2D for 10 days and in 3D for short-terms (~2 weeks) or long-terms (1 month) in vitro (Figure 1A). We monitored stable pBMAd presence in 2D transwells via Cell TiterGlo and on 3D scaffolds via RealTime Glo up to 9 days post-seeding, and by calcein staining after 10 days in culture. Using bioluminescence in pBMAs (Cell TiterGlo) and MM.1S (luciferin spike-in) we were able to detect viability of both cell types in co-culture. Utilizing mass spectrometry proteomics, in pBMAds alone, we detected secretion of CXCL12 (SDF1; p<0.01), as well as TGFB1 (p<0.01) which has implications for bone remodeling. Interestingly, in conditioned media collected from the co-culture condition, we found increased secretion of the WNT inhibitor DKK3 (p<0.05) and detection of proteins known to be associated with extracellular exosomes including: CNTN1, ACP1, OMD, RPS18, RPL24, PGAM1, LAMA4, CTSB, PLOD2 and ARHGDIA (Figure 1B), suggesting that pBMAds and MM cells likely interact via exosomes. Conclusions: In sum, we developed three in vitro cell culture systems to study primary bone marrow adipocytes and myeloma cells, which could be adapted to investigate many diseases and biological processes involving the bone marrow, including other bone-homing tumor types. Our use of these models to examine the interaction between MM cells and pBMAds has revealed new roles for pBMAds in the MM bone marrow microenvironment, with implications for MM-induced bone disease and pBMAd-MM bidirectional signaling. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Left Atrial Structural and Functional Response in Kidney Transplant Recipients Treated With Mesenchymal Stromal Cell Therapy and Early Tacrolimus Withdrawal

Autologous bone marrow-derived mesenchymal stromal cell (MSC) therapy and withdrawal of calcineurin inhibitors (CNIs) has been shown to improve systemic blood pressure control and left ventricular hypertrophy regression in kidney transplant recipients. In the current subanalysis, we aimed to evaluate the impact of this novel immunosuppressive regimen on the longitudinal changes of left atrial (LA) structure and function after kidney transplantation. Kidney transplant recipients randomized to MSC therapy-infused at weeks 6 and 7 after transplantation, with complete discontinuation at week 8 of tacrolimus (MSC group)-or standard tacrolimus dose (control group) were evaluated with transthoracic echocardiography at weeks 4 and 24 after kidney transplantation. The changes in echocardiographic parameters were compared between the randomization arms using an analysis of covariance model adjusted for baseline variable. Fifty-four participants (MSC therapy=27; tacrolimus therapy=27) were included. There was no significant interaction between the allocated treatment and the changes of indexed maximal LA volume (LAVImax) over the study period. Conversely, between 4 and 24weeks post-transplantation, an increase in indexed minimal LA volume (LAVImin) was observed in control subjects, while it remained unchanged in the MSC group, leading to a significant difference between groups (P=.021). Additionally, patients treated with MSC therapy showed a benefit in LA function, assessed by a significant interaction between changes in LA emptying fraction and LA reservoir strain and the randomization arm (P=.012 and P=.027, respectively). The combination of MSC therapy and CNIs withdrawal prevents progressive LA dilation and dysfunction in the first 6months after kidney transplantation. LAVImin and LA reservoir strain may be more sensitive markers of LA reverse remodeling, compared with LAVImax.

Proteomics profile of mesenchymal stromal cells and extracellular vesicles in normoxic and hypoxic conditions

Background aimsAlthough bone marrow-derived mesenchymal stromal cells (MSCs) have demonstrated success in pre-clinical studies, they have shown only mild therapeutic effects in clinical trials. Hypoxia pre-conditioning may optimize the performance of bone marrow-derived MSCs because it better reflects the physiological conditions of their origin. It is not known whether changes in the protein profile caused by hypoxia in MSCs can be extended to the extracellular vesicles (EVs) released from them. The aim of this study was to evaluate the proteomics profile of MSCs and their EVs under normoxic and hypoxic conditions. MethodsBone marrow-derived MSCs were isolated from six healthy male Wistar rats. After achieving 80% confluence, MSCs were subjected to normoxia (MSC-Norm) (21% oxygen, 5% carbon dioxide, 74% nitrogen) or hypoxia (MSC-Hyp) (1% oxygen, 5% carbon dioxide, 94% nitrogen) for 48 h. Cell viability and oxygen consumption rate were assessed. EVs were extracted from MSCs for each condition (EV-Norm and EV-Hyp) by ultracentrifugation. Total proteins were isolated from MSCs and EVs and prepared for mass spectrometry. EVs were characterized by nanoparticle tracking analysis. Proteomics data were analyzed by PatternLab 4.0, Search Tool for the Retrieval of Interacting Genes/Proteins, Gene Ontology, MetaboAnalyst and Reactome software. ResultsCell viability was higher in MSC-Hyp than MSC-Norm (P = 0.007). Basal respiration (P = 0.001), proton leak (P = 0.004) and maximal respiration (P = 0.014) were lower in MSC-Hyp than MSC-Norm, and no changes in adenosine triphosphate-linked and residual respiration were observed. The authors detected 2177 proteins in MSC-Hyp and MSC-Norm, of which 147 were identified in only MSC-Hyp and 512 were identified in only MSC-Norm. Furthermore, 718 proteins were identified in EV-Hyp and EV-Norm, of which 293 were detected in only EV-Hyp and 30 were detected in only EV-Norm. Both MSC-Hyp and EV-Hyp showed enrichment of pathways and biological processes related to glycolysis, the immune system and extracellular matrix organization. ConclusionsMSCs subjected to hypoxia showed changes in their survival and metabolic activity. In addition, MSCs under hypoxia released more EVs, and their content was related to expression of regulatory proteins of the immune system and extracellular matrix organization. Because of the upregulation of proteins involved in glycolysis, gluconeogenesis and glucose uptake during hypoxia, production of reactive oxygen species and expression of immunosuppressive properties may be affected.

An In Vitro Model to Test the Influence of Immune Cell Secretome on Mesenchymal Stromal Cell Osteogenic Differentiation.

Immune cells and their soluble factors have an important role in the bone healing process. Modulation of the immune response, therefore, offers a potential strategy to enhance bone formation. To investigate the influence of the immune system on osteogenesis, we developed and applied an in vitro model that incorporates both innate and adaptive immune cells. Human peripheral blood mononuclear cells (PBMCs) were isolated and cultured for 24 h and subsequently stimulated with immune-modulatory agents; C-class CpG oligodeoxynucleotide (CpG ODN C), polyinosinic acid-polycytidylic acid [Poly(I:C)], and lipopolysaccharide (LPS); all pathogen recognition receptor agonists, that target Toll-like receptors (TLRs) 9, 3, and 4, respectively. The conditioned medium (CM) obtained from PBMCs after 24 h was used to investigate its effects on the metabolic activity and osteogenic differentiation capacity of human bone marrow-derived mesenchymal stromal cells (MSCs). Conditioned media from unstimulated PBMCs did not affect the metabolic activity and osteogenic differentiation capacity of MSCs. The CM from CpG ODN C and LPS-stimulated PBMCs increased alkaline phosphatase activity (ALP) of MSCs by approximately threefold as compared with the unstimulated control, whereas Poly(I:C) CM did not enhance ALP activity of MSCs. Moreover, direct stimulation of MSCs with the immune-modulatory stimuli did not result in increased ALP. These results demonstrate that soluble factors present in CM from PBMCs stimulated with immune-modulatory factors enhance osteogenesis of MSCs. This in vitro model can serve as a tool in screening immune-modulatory stimulants from a broad variety of immune cells for (indirect) effects on osteogenesis and also to identify soluble factors from multiple immune cell types that may modulate bone healing.

The Effects of the Combination of Mesenchymal Stromal Cells and Nanofiber-Hydrogel Composite on Repair of the Contused Spinal Cord.

A bone marrow-derived mesenchymal stromal cell (MSC) transplant and a bioengineered nanofiber-hydrogel composite (NHC) have been shown to stimulate nervous tissue repair in the contused spinal cord in rodent models. Here, these two modalities were combined to assess their repair effects in the contused spinal cord in adult rats. Cohorts of contused rats were treated with MSC in NHC (MSC-NHC), MSC in phosphate-buffered saline (MSC-PBS), NHC, or PBS injected into the contusion site at 3 days post-injury. One week after injury, there were significantly fewer CD68+ cells in the contusion with MSC-NHC and NHC, but not MSC-PBS. The reduction in CD86+ cells in the injury site with MSC-NHC was mainly attributed to NHC. One and eight weeks after injury, we found a greater CD206+/CD86+ cell ratio with MSC-NHC or NHC, but not MSC-PBS, indicating a shift from a pro-inflammatory towards an anti-inflammatory milieu in the injury site. Eight weeks after injury, the injury size was significantly reduced with MSC-NHC, NHC, and MSC-PBS. At this time, astrocyte, and axon presence in the injury site was greater with MSC-NHC compared with MSC-PBS. We did not find a significant effect of NHC on MSC transplant survival, and hind limb function was similar across all groups. However, we did find fewer macrophages at 1 week post-injury, more macrophages polarized towards a pro-regenerative phenotype at 1 and 8 weeks after injury, and reduced injury volume, more astrocytes, and more axons at 8 weeks after injury in rats with MSC-NHC and NHC alone compared with MSC-PBS; these findings were especially significant between rats with MSC-NHC and MSC-PBS. The data support further study in the use of an NHC-MSC combination transplant in the contused spinal cord.

Assessment of the LOVO device for final harvest of novel cell therapies: a Production Assistance for Cellular Therapies multi-center study

Background aimsThe final harvest or wash of a cell therapy product is an important step in manufacturing, as viable cell recovery is critical to the overall success of a cell therapy. Most harvest/wash approaches in the clinical lab involve centrifugation, which can lead to loss of cells and decreased viability of the final product. Here the authors report on a multi-center assessment of the LOVO Cell Processing System (Fresenius Kabi, Bad Homburg, Germany), a cell processing device that uses a spinning filtration membrane instead of centrifugation. MethodsFour National Institutes of Health Production Assistance for Cellular Therapies cell processing facilities (CPFs) assessed the LOVO Cell Processing System for final harvest and/or wash of the following three different cell products: activated T cells (ATCs), tumor-infiltrating lymphocytes (TILs) and bone marrow-derived mesenchymal stromal cells (MSCs). Each site compared their current in-house, routinely used method of final cell harvest and/or wash with that of the LOVO device. ResultsFinal harvest and/or wash of ATCs, TILs and MSCs using the LOVO system resulted in satisfactory cell viability and recovery with some substantial improvement over the in-house methods of CPFs. Processing time was variable among cell types/facilities. ConclusionsThe LOVO Cell Processing System provides an alternative to centrifuge-based technologies. The system employs a spinning membrane filter, exposing cells to minimal g-forces compared with centrifugation, and is automated and closed. This small multi-center study demonstrated the ability of the LOVO device to yield satisfactory cell viability and recovery of T cells and MSCs.

Bone Marrow Multipotent Mesenchymal Stromal Cells from Patients with Aplastic Anemia Retain Their Ability to Support Hematopoietic Precursors despite Pronounced Changes in Gene Expression.

The properties of bone marrow-derived multipotent mesenchymal stromal cells (MSC) of patients with aplastic anemia at the onset of the disease are studied insufficiently. The aim of this work was to test the ability of MSC from patients with aplastic anemia to maintain hematopoietic precursors and to analyze the expression of genes associated with hematopoiesis and immune response. The ability of MSC to maintain hematopoietic precursors was determined by counting cobblestone area-forming cells; gene expression was analyzed by quantitative PCR. It was shown that MSC of patients with aplastic anemia preserve their ability to maintain hematopoietic precursors. Pronounced changes in the expression of the VEGFA and ANGPT1 genes were found. MSC from aplastic anemia patients with PNH clone significantly differ from those from aplastic anemia patients without PNH clone in terms of the expression of the SDF1, IL1R, and VEGFA genes. Changes in gene expression can be associated with the pathogenesis of the disease.

Reduced expression of mitochondrial fumarate hydratase in progressive multiple sclerosis contributes to impaired in vitro mesenchymal stromal cell-mediated neuroprotection

Background: Cell-based therapies for multiple sclerosis (MS), including those employing autologous bone marrow-derived mesenchymal stromal cells (MSC) are being examined in clinical trials. However, recent studies have identified abnormalities in the MS bone marrow microenvironment. Objective: We aimed to compare the secretome of MSC isolated from control subjects (C-MSC) and people with MS (MS-MSC) and explore the functional relevance of findings. Methods: We employed high throughput proteomic analysis, enzyme-linked immunosorbent assays and immunoblotting, as well as in vitro assays of enzyme activity and neuroprotection. Results: We demonstrated that, in progressive MS, the MSC secretome has lower levels of mitochondrial fumarate hydratase (mFH). Exogenous mFH restores the in vitro neuroprotective potential of MS-MSC. Furthermore, MS-MSC expresses reduced levels of fumarate hydratase (FH) with downstream reduction in expression of master regulators of oxidative stress. Conclusions: Our findings are further evidence of dysregulation of the bone marrow microenvironment in progressive MS with respect to anti-oxidative capacity and immunoregulatory potential. Given the clinical utility of the fumaric acid ester dimethyl fumarate in relapsing–remitting MS, our findings have potential implication for understanding MS pathophysiology and personalised therapeutic intervention.

Human Liver Stem Cell-Derived Extracellular Vesicles Target Hepatic Stellate Cells and Attenuate Their Pro-fibrotic Phenotype.

Liver fibrosis occurs in response to chronic liver injury and is characterized by an excessive deposition of extracellular matrix. Activated hepatic stellate cells are primarily responsible for this process. A possible strategy to counteract the development of hepatic fibrosis could be the reversion of the activated phenotype of hepatic stellate cells. Extracellular vesicles (EVs) are nanosized membrane vesicles involved in intercellular communication. Our previous studies have demonstrated that EVs derived from human liver stem cells (HLSCs), a multipotent population of adult stem cells of the liver with mesenchymal-like phenotype, exert in vivo anti-fibrotic activity in the liver. However, the mechanism of action of these EVs remains to be determined. We set up an in vitro model of hepatic fibrosis using a human hepatic stellate cell line (LX-2) activated by transforming growth factor-beta 1 (TGF-β1). Then, we investigated the effect of EVs obtained from HLSCs and from human bone marrow-derived mesenchymal stromal cells (MSCs) on activated LX-2. The incubation of activated LX-2 with HLSC-EVs reduced the expression level of alpha-smooth muscle actin (α-SMA). Conversely, MSC-derived EVs induced an increase in the expression of pro-fibrotic markers in activated LX-2. The analysis of the RNA cargo of HLSC-EVs revealed the presence of several miRNAs involved in the regulation of fibrosis and inflammation. Predictive target analysis indicated that several microRNAs (miRNAs) contained into HLSC-EVs could possibly target pro-fibrotic transcripts. In particular, we demonstrated that HLSC-EVs shuttled miR-146a-5p and that treatment with HLSC-EVs increased miR-146a-5p expression in LX-2. In conclusion, this study demonstrates that HLSC-EVs can attenuate the activated phenotype of hepatic stellate cells and that their biological effect may be mediated by the delivery of anti-fibrotic miRNAs, such as miR-146a-5p.

Determining minimally clinically important differences for outcome measures in patients with chronic motor deficits secondary to traumatic brain injury

ABSTRACT Objective To determine minimally clinically important differences (MCIDs) for Disability Rating Scale (DRS), Fugl-Meyer Upper Extremity Subscale (FM-UE), Fugl-Meyer Lower Extremity Subscale (FM-LE), and Fugl-Meyer Motor Scale (FMMS) in patients with chronic motor deficits secondary to traumatic brain injury (TBI). Methods Retrospective analysis from the 1-year, double-blind, randomized, surgical sham-controlled, Phase 2 STEMTRA trial (NCT02416492), in which patients with chronic motor deficits secondary to TBI (N = 61) underwent intracerebral stereotactic implantation of modified bone marrow-derived mesenchymal stromal (SB623) cells. MCIDs for DRS, FM-UE, FM-LE, and FMMS were triangulated with distribution-based, anchor-based, and Delphi panel estimates. Results Triangulated MCIDs were: 1) −1.5 points for the Disability Rating Scale; 2) 6.2 points for the Fugl-Meyer Upper Extremity Subscale; 3) 3.2 points for the Fugl-Meyer Lower Extremity Subscale; and 4) 8.4 points for the Fugl-Meyer Motor Scale. Conclusions For the first time in the setting of patients with chronic motor deficits secondary to TBI, this study reports triangulated MCIDs for: 1) DRS, a measure of global outcome; and 2) Fugl-Meyer Scales, measures of motor impairment. These findings guide the use of DRS and Fugl-Meyer Scales in the assessment of global disability outcome and motor impairment in future TBI clinical trials.