Number Of Mesenchymal Stromal Cells Research Articles

Dose-dependent effect of mesenchymal stromal cells co-grafted with dopaminergic neurons in a Parkinson's disease rat model.

A major limiting factor for cell therapy in Parkinson's disease is the poor survival and reinnervation capacity of grafted dopaminergic neurons, independently of the cell source. Mesenchymal stromal cells (MSCs) have high capability to regulate the local environment through the release of trophic, antiapoptotic and immunomodulatory factors. In this work, we investigated whether co‐grafting of MSCs could improve the survival and reinnervation ability of dopaminergic precursors transplanted in animal models of Parkinson's disease. Rats with total unilateral dopaminergic denervation were grafted with a cell suspension of rat dopaminergic precursors (500,000 cells) with or without a high (200,000 cells) or low (25,000 cells) number of MSCs. Eight weeks after grafting, rats were tested for motor behaviour and sacrificed for histological analysis. Our results showed that the survival of dopaminergic neurons and graft‐derived striatal dopaminergic innervation was higher in rats that received co‐grafts containing a low number of MSCs than in non‐co‐grafted controls. However, the survival of dopaminergic neurons and graft‐derived dopaminergic reinnervation was lower in rats receiving co‐grafts with high number of MSCs than in non‐co‐grafted controls. In conclusion, co‐grafting with MSCs or MSCs‐derived products may constitute a useful strategy to improve dopaminergic graft survival and function. However, a tight control of MSCs density or levels of MSCs‐derived products is necessary.

Systemic Administration of G-CSF Accelerates Bone Regeneration and Modulates Mobilization of Progenitor Cells in a Rat Model of Distraction Osteogenesis.

Granulocyte colony-stimulating factor (G-CSF) was shown to promote bone regeneration and mobilization of vascular and osteogenic progenitor cells. In this study, we investigated the effects of a systemic low dose of G-CSF on both bone consolidation and mobilization of hematopoietic stem/progenitor cells (HSPCs), endothelial progenitor cells (EPCs) and mesenchymal stromal cells (MSCs) in a rat model of distraction osteogenesis (DO). Neovascularization and mineralization were longitudinally monitored using positron emission tomography and planar scintigraphy. Histological analysis was performed and the number of circulating HSPCs, EPCs and MSCs was studied by flow cytometry. Contrary to control group, in the early phase of consolidation, a bony bridge with lower osteoclast activity and a trend of an increase in osteoblast activity were observed in the distracted callus in the G-CSF group, whereas, at the late phase of consolidation, a significantly lower neovascularization was observed. While no difference was observed in the number of circulating EPCs between control and G-CSF groups, the number of MSCs was significantly lower at the end of the latency phase and that of HSPCs was significantly higher 4 days after the bone lengthening. Our results indicate that G-CSF accelerates bone regeneration and modulates mobilization of progenitor cells during DO.

Splenic macrophage phagocytosis of intravenously infused mesenchymal stromal cells attenuates tumor localization

Mesenchymal stromal cells (MSCs) possess remarkable tumor tropism, making them ideal vehicles to deliver tumor-targeted therapeutic agents; however, their value in clinical medicine has yet to be realized. A barrier to clinical utilization is that only a small fraction of infused MSCs ultimately localize to the tumor. In an effort to overcome this obstacle, we sought to enhance MSC trafficking by focusing on the factors that govern MSC arrival within the tumor microenvironment. Our findings show that MSC chemoattraction is only present in select tumors, including osteosarcoma, and that the chemotactic potency among similar tumors varies substantially. Using an osteosarcoma xenograft model, we show that human MSCs traffic to the tumor within several hours of infusion. After arrival, MSCs are observed to localize in clusters near blood vessels and MSC-associated bioluminescence signal intensity is increased, suggesting that the seeded cells expand after engraftment. However, our studies reveal that a significant portion of MSCs are eliminated en route by splenic macrophage phagocytosis, effectively limiting the number of cells available for tumor engraftment. To increase MSC survival, we transiently depleted macrophages with liposomal clodronate, which resulted in increased tumor localization without substantial reduction in tumor-associated macrophages. Our data suggest that transient macrophage depletion will significantly increase the number of MSCs in the spleen and thus improve MSC localization within a tumor, theoretically increasing the effective dose of an anti-cancer agent. This strategy may subsequently improve the clinical efficacy of MSCs as vehicles for the tumor-directed delivery of therapeutic agents.

Mesenchymal stromal cell treatment of donor kidneys during ex vivo normothermic machine perfusion: A porcine renal autotransplantation study.

Normothermic machine perfusion (NMP) of injured kidneys offers the opportunity for interventions to metabolically active organs prior to transplantation. Mesenchymal stromal cells (MSCs) can exert regenerative and anti-inflammatory effects in ischemia-reperfusion injury. The aims of this study were to evaluate the safety and feasibility of MSC treatment of kidneys during NMP using a porcine autotransplantation model, and examine potential MSC treatment-associated kidney improvements up to 14days posttransplant. After 75min of kidney warm ischemia, four experimental groups of n=7 underwent 14h of oxygenated hypothermic machine perfusion. In three groups this was followed by 240min of NMP with infusion of vehicle, 10million porcine, or 10million human adipose-derived MSCs. All kidneys were autotransplanted after contralateral nephrectomy. MSC treatment did not affect perfusion hemodynamics during NMP or cause adverse effects at reperfusion, with 100% animal survival. MSCs did not affect plasma creatinine, glomerular filtration rate, neutrophil gelatinase-associated lipocalin concentrations or kidney damage assessed by histology during the 14days, and MSCs retention was demonstrated in renal cortex. Infusing MSCs during ex vivo NMP of porcine kidneys was safe and feasible. Within the short posttransplant follow-up period, no beneficial effects of ex vivo MSC therapy could be demonstrated.

Interactions of human mesenchymal stromal cells with peripheral blood mononuclear cells in a Mitogenic proliferation assay

BackgroundImmunomodulation by mesenchymal stromal cells (MSCs) is a potentially important therapeutic modality. MSCs suppress peripheral blood mononuclear cell (PBMC) proliferation in vitro, suggesting a mechanism for suppressing inflammatory responses in vivo. This study details the interactions of PBMCs and MSCs. MethodsPooled human PBMCs and MSCs were co-cultured at different MSC:PBMC ratios and harvested from 0 to 120 h, with and without phytohaemagglutin A (PHA) stimulation. Proliferation of adherent MSCs and non-adherent PBMCs was assessed by quantitation of ATP levels. PBMC surface marker expression was analyzed by flow cytometry. Indoleamine 2,3-dioxygenase (IDO) activity was determined by kynurenine assay and IDO mRNA by RT-PCR. Cytokine release was measured by ELISA. Immunofluorescent microscopy detected MSC, PBMC, monocyte (CD14+) and apoptotic events. ResultsPBMC proliferation in response to PHA gave a robust ATP signal by 72 h, which was suppressed by co-culture with densely plated MSCs. Very low level MSC seeding densities relative to PBMC number reproducibly stimulated PBMC proliferation. The CD4+/CD3+ population significantly decreased over time while the CD8+/CD3+ population significantly increased. No change in CD4+/CD8+ ratio is seen with high density MSC co-culture; very low density MSCs augment the changes seen in PHA stimulated PBMCs alone. IDO activity in MSCs co-cultured with PBMCs correlated with PBMC suppression. MSCs increased the secretion of IL-10 and IL-6 from stimulated co-cultures and decreased TNF-α secretion. In stimulated co-culture, low density MSCs decreased in number; fluorescence immunomicroscopy detected association of PBMC with MSC and phosphatidyl serine externalization in both cell populations. ConclusionsA bidirectional interaction between MSCs and PBMCs occurs during co-culture. High numbers of MSCs inhibit PHA-stimulated PBMC proliferation and the PBMC response to stimulation; low numbers of MSCs augment these responses. Low density MSCs are susceptible to attrition, apparently by PBMC-induced apoptosis. These results may have direct application when considering therapeutic dosing of patients; low MSC doses may have unintended detrimental consequences.

Inhibition of longevity regulator PAPP-A modulates tissue homeostasis via restraint of mesenchymal stromal cells.

Pregnancy‐associated plasma protein‐A (PAPP‐A) is a secreted metalloprotease that increases insulin‐like growth factor (IGF) availability by cleaving IGF‐binding proteins. Reduced IGF signaling extends longevity in multiple species, and consistent with this, PAPP‐A deletion extends lifespan and healthspan; however, the mechanism remains unclear. To clarify PAPP‐A’s role, we developed a PAPP‐A neutralizing antibody and treated adult mice with it. Transcriptomic profiling across tissues showed that anti‐PAPP‐A reduced IGF signaling and extracellular matrix (ECM) gene expression system wide. The greatest reduction in IGF signaling occurred in the bone marrow, where we found reduced bone, marrow adiposity, and myelopoiesis. These diverse effects led us to search for unifying mechanisms. We identified mesenchymal stromal cells (MSCs) as the source of PAPP‐A in bone marrow and primary responders to PAPP‐A inhibition. Mice treated with anti‐PAPP‐A had reduced IGF signaling in MSCs and dramatically decreased MSC number. As MSCs are (1) a major source of ECM and the progenitors of ECM‐producing fibroblasts, (2) the originating source of adult bone, (3) regulators of marrow adiposity, and (4) an essential component of the hematopoietic niche, our data suggest that PAPP‐A modulates bone marrow homeostasis by potentiating the number and activity of MSCs. We found that MSC‐like cells are the major source of PAPP‐A in other tissues also, suggesting that reduced MSC‐like cell activity drives the system‐wide reduction in ECM gene expression due to PAPP‐A inhibition. Dysregulated ECM production is associated with aging and drives age‐related diseases, and thus, this may be a mechanism by which PAPP‐A deficiency enhances longevity.

G-CSF treatment of healthy pediatric donors affects their hematopoietic microenvironment through changes in bone marrow plasma cytokines and stromal cells

Although G-CSF mobilized peripheral blood stem cell (PBSC) transplantation is commonly used in adults, bone marrow (BM) is still the preferred stem cell source in pediatric stem cell transplantation. Despite the fact that G-CSF is increasingly being used to enhance the hematopoietic stem/progenitor cell (HSPC) yield in BM transplantation (G-BM), the direct effects of G-CSF on the pediatric BM microenvironment have never been investigated. The BM hematopoietic niche provides the physical space where the HSPCs reside. This BM niche regulates HSPC quiescence and proliferation through direct interactions with other niche cells, including Mesenchymal Stromal Cells (MSCs). These cells have been shown to secrete a wide range of hematopoietic cytokines (CKs) and growth factors (GFs) involved in differentiation, retention and homing of hematopoietic cells. Here, we assessed changes in the BM microenvironment by measuring levels of 48 different CKs and GFs in G-BM and control BM (C-BM) plasma from pediatric donors. In addition, the effect of G-CSF on cell numbers and characteristics of HSPCs and MSCs was assessed. IL-16, SCGF-b, MIP-1b (all >1000 pg/mL) and RANTES (>10.000 pg/mL) were highly expressed in healthy donor pediatric BM plasma. Levels of IL-3, IL-18, GROa, MCP-3 (p<0.05) were increased in G-BM, whereas levels of RANTES (p<0.001) decreased after G-CSF treatment. We found a negative correlation with increasing age for IL2-Ra and LIF (p<0.05). In addition, a concomitant increase in the number of both hematopoietic and fibroblast colony forming units was observed, indicating that G-CSF affects both HSPC and MSC numbers. In conclusion, G-CSF treatment of healthy pediatric donors affects the hematopoietic BM microenvironment by expansion of HSPC and MSC numbers and modifying local CK and GF levels.

Hypoxia-cultured mouse mesenchymal stromal cells from bone marrow and compact bone display different phenotypic traits

It has been suggested that the bone marrow microenvironment harbors two distinct populations of mesenchymal stromal cells (MSC), one with a perivascular location and other present in the endosteum. A better understanding of the biology of these MSC subsets has been pursued in order to refine its clinical application. However, most comparative characterizations of mouse MSC have been performed in normoxia. This can result in misleading interpretations since mouse MSC subsets with low/defective p53 activity are known to be selected during culture in normoxia. Here, we report a comprehensive in vitro characterization of mouse MSC isolated from bone marrow (BM-MSC) and compact bone (CB-MSC) expanded and assayed under hypoxia for their morphology, clonogenic efficiency and differentiation capacity. We found that, under hypoxia, compact bone is richer in absolute numbers of MSC and isolation of MSC from compact bone is associated with a reduced risk of hematopoietic cell carryover. In addition, CB-MSC have higher in vitro osteogenic capacity than BM-MSC, while adipogenic differentiation potential is similar. These findings reinforce the hypothesis of the existence of MSC in bone marrow and compact bone representing functionally distinct cell populations and highlight the compact bone as an efficient source of murine MSC under physiological oxygen concentrations.

Tolerance to Bone Marrow Transplantation: Do Mesenchymal Stromal Cells Still Have a Future for Acute or Chronic GvHD?

Mesenchymal Stromal Cells (MSCs) are fibroblast-like cells of mesodermal origin present in many tissues and which have the potential to differentiate to osteoblasts, adipocytes and chondroblasts. They also have a clear immunosuppressive and tissue regeneration potential. Indeed, the initial classification of MSCs as pluripotent stem cells, has turned into their identification as stromal progenitors. Due to the relatively simple procedures available to expand in vitro large numbers of GMP grade MSCs from a variety of different tissues, many clinical trials have tested their therapeutic potential in vivo. One pathological condition where MSCs have been quite extensively tested is steroid resistant (SR) graft versus host disease (GvHD), a devastating condition that may occur in acute or chronic form following allogeneic hematopoietic stem cell transplantation. The clinical and experimental results obtained have outlined a possible efficacy of MSCs, but unfortunately statistical significance in clinical studies has only rarely been reached and effects have been relatively limited in most cases. Nonetheless, the extremely complex pathogenetic mechanisms at the basis of GvHD, the fact that studies have been conducted often in patients who had been previously treated with multiple lines of therapy, the variable MSC doses and schedules administered in different trials, the lack of validated potency assays and clear biomarkers, the difference in MSC sources and production methods may have been major factors for this lack of clear efficacy in vivo. The heterogeneity of MSCs and their different stromal differentiation potential and biological activity may be better understood through more refined single cell sequencing and proteomic studies, where either an “anti-inflammatory” or a more “immunosuppressive” profile can be identified. We summarize the pathogenic mechanisms of acute and chronic GvHD and the role for MSCs. We suggest that systematic controlled clinical trials still need to be conducted in the most promising clinical settings, using better characterized cells and measuring efficacy with specific biomarkers, before strong conclusions can be drawn about the therapeutic potential of these cells in this context. The same analysis should be applied to other inflammatory, immune or degenerative diseases where MSCs may have a therapeutic potential.

The Metastatic Bone Marrow Niche in Neuroblastoma: Altered Phenotype and Function of Mesenchymal Stromal Cells.

Simple SummaryPatients with metastatic neuroblastoma are at risk for relapse after conventional therapy. It is believed that residual neuroblastoma cells in the bone marrow (BM) are responsible for this process after becoming resistant to chemotherapy. Studies in various tumor types suggest that cells in the BM environment (specifically the so-called mesenchymal stromal cells (MSCs)) can promote survival and chemoresistance of tumor cells. We characterized the MSC-compartment within the BM of patients with neuroblastoma, with and without metastases. Our data indicate that the quantity and functionality of the MSC population is altered in the presence of tumor cells. We furthermore identify a specific MSC-subpopulation which is exclusively detected in the presence of tumor cells. These data contribute to the understanding of the interactions between neuroblastoma cells and the environment, which is essential in order to target the tumor cells in the BM more effectively, thereby improving therapy success and preventing relapse.Background: The bone marrow (BM) is the main site of metastases and relapse in patients with neuroblastoma (NB). BM-residing mesenchymal stromal cells (MSCs) were shown to promote tumor cell survival and chemoresistance. Here we characterize the MSC compartment of the metastatic NB BM niche. Methods: Fresh BM of 62 NB patients (all stages), and control fetal and adult BM were studied by flow cytometry using well-established MSC-markers (CD34−, CD45−, CD90+, CD105+), and CD146 and CD271 subtype-markers. FACS-sorted BM MSCs and tumor cells were validated by qPCR. Moreover, isolated MSCs were tested for multilineage differentiation and Colony-forming-unit-fibroblasts (CFU-Fs) capacity. Results: Metastatic BM contains a higher number of MSCs (p < 0.05) with increased differentiation capacity towards the osteoblast lineage. Diagnostic BM contains a MSC-subtype (CD146+CD271−), only detected in BM of patients with metastatic-NB, determined by flow cytometry. FACS-sorting clearly discriminated MSC(-subtypes) and NB fractions, validated by mRNA and DNA qPCR. Overall, the CD146+CD271− subtype decreased during therapy and was detected again in the majority of patients at relapse. Conclusions: We demonstrate that the neuroblastoma BM-MSC compartment is different in quantity and functionality and contains a metastatic-niche-specific MSC-subtype. Ultimately, the MSCs contribution to tumor progression could provide targets with potential for eradicating resistant metastatic disease.

Effects of Conditioned Medium from Bone Marrow Cells on Human Umbilical Cord Perivascular Cells.

Mesenchymal cells derived from human umbilical cord tissue are attracting increasing attention as a source for cell therapy. However, for applying the same in tissue engineering, it has been shown that the differentiation capacity of mesenchymal stromal cells (MSCs) is influenced by the tissue from which the cells are harvested. Thus, to explore the possibility of increasing the osteogenic capacity of MSCs derived from the perivascular tissue of the human umbilical cord (human umbilical cord perivascular cells, HUCPVCs), we cultured these cells using conditioned medium (CM) derived from cultures of human bone marrow-derived mesenchymal stromal cells (hBMMSCs). However, hBM-CM contains a wide variety of growth factors, the amounts and ratios of which are considered to vary with the cell culture stage. Thus, we aimed to evaluate the effects of hBM-CM derived from different stages of hBMMSC culture on the osteogenic capacity of HUCPVCs. The stages of hBMMSC culture were defined as follows: Stage 1 (mitogenic stage) represented the period from the start of hBMMSC culture to 70% cell confluence; Stage 2 (confluent stage) represented the period from 70% confluence to the initiation of calcified nodule formation; and Stage 3 (calcification stage) represented the period following the initiation of calcified nodule formation. An analysis of growth factors contained in the CM obtained at each stage by enzyme-linked immunosorbent assay showed that insulin-like growth factor 1 (IGF-1) was significantly elevated at Stage 2, whereas vascular endothelial growth factor (VEGF) was significantly elevated at Stage 3. HUCPVCs were cultured using the CM from each of the stages for 1, 2, or 3 weeks. RUNX2 expression was the most upregulated at week 1 and then downregulated in all the groups. The expression of collagen 1 was significantly elevated in Stage 2 HUCs at week 3. Alkaline phosphatase (ALP) activity, ALP, and alizarin staining were higher in Stage 2 HUCs and Stage 3 HUCs. The calcium content was the highest in Stage 2 HUCs. The calcium content of HUCPVC obtained by the method used in this study was six times higher than that reported in the previous study. Collectively, our results show that the CM obtained at Stage 2 was most effective in driving the osteogenic differentiation of HUCPVCs. Impact Statement Mesenchymal stromal cells (MSCs) derived from the perivascular tissue of umbilical cords are promising candidates for regenerative medicine. Because these are able to be differentiated into bone cells, cartilage cells, and adipocytes. The number of MSCs in perivascular tissue (HUCPVCs) is ∼1/300 but the number of HUCPVCs that differentiates into osteogenic cells is quite low. In order to promote osteogenic differentiation of HUCPVCs, we cultured HUCPVCs using conditioned medium collected from human bone marrow-derived mesenchymal stromal cells. Our study suggests that the use of conditioned medium can be effective on inducing osteogenic differentiation of HUCPVCs.

A Xeno-Free Strategy for Derivation of Human Umbilical Vein Endothelial Cells and Wharton's Jelly Derived Mesenchymal Stromal Cells: A Feasibility Study toward Personal Cell and Vascular Based Therapy.

Coimplantation of endothelial cells (ECs) and mesenchymal stromal cells (MSCs) into the transplantation site could be a feasible option to achieve a sufficient level of graft-host vascularization. To find a suitable source of tissue that provides a large number of high-quality ECs and MSCs suited for future clinical application, we developed a simplified xeno-free strategy for isolation of human umbilical vein endothelial cells (HUVECs) and Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs) from the same umbilical cord. We also assessed whether the coculture of HUVECs and WJ-MSCs derived from the same umbilical cord (autogenic cell source) or from different umbilical cords (allogenic cell sources) had an impact on in vitro angiogenic capacity. We found that HUVECs grown in 5 ng/ml epidermal growth factor (EGF) supplemented xeno-free condition showed higher proliferation potential compared to other conditions. HUVECs and WJ-MSCs obtained from this technic show an endothelial lineage (CD31 and von Willebrand factor) and MSC (CD73, CD90, and CD105) immunophenotype characteristic with high purity, respectively. It was also found that only the coculture of HUVEC/WJ-MSC, but not HUVEC or WJ-MSC mono-culture, provides a positive effect on vessel-like structure (VLS) formation, in vitro. Further investigations are needed to clarify the pros and cons of using autogenic or allogenic source of EC/MSC in tissue engineering applications. To the best of our knowledge, this study offers a simple, but reliable, xeno-free strategy to establish ECs and MSCs from the same umbilical cord, a new opportunity to facilitate the development of personal cell-based therapy.

Ceramic Scaffolds in a Vacuum Suction Handle for Intraoperative Stromal Cell Enrichment.

During total joint replacement, high concentrations of mesenchymal stromal cells (MSCs) are released at the implantation site. They can be found in cell–tissue composites (CTC) that are regularly removed by surgical suction. A surgical vacuum suction handle was filled with bone substitute granules, acting as a filter allowing us to harvest CTC. The purpose of this study was to investigate the osteopromotive potential of CTC trapped in the bone substitute filter material during surgical suction. In the course of 10 elective total hip and knee replacement surgeries, β-tricalcium-phosphate (TCP) and cancellous allograft (Allo) were enriched with CTC by vacuum suction. Mononuclear cells (MNC) were isolated from the CTC and investigated towards cell proliferation and colony forming unit (CFU) formation. Furthermore, MSC surface markers, trilineage differentiation potential and the presence of defined cytokines were examined. Comparable amounts of MNC and CFUs were detected in both CTCs and characterized as MSC‰ of MNC with 9.8 ± 10.7‰ for the TCP and 12.8 ± 10.2‰ for the Allo (p = 0.550). CTCs in both filter materials contain cytokines for stimulation of cell proliferation and differentiation (EGF, PDGF-AA, angiogenin, osteopontin). CTC trapped in synthetic (TCP) and natural (Allo) bone substitute filters during surgical suction in the course of a joint replacement procedure include relevant numbers of MSCs and cytokines qualified for bone regeneration.

Molecular and morphological peculiarities of chronic placental insufficiency formation caused by different types of diabetes mellitus

Рathogenesis of the chronic placental insufficiency is largely determined by the type of diabetes mellitus and the degree of its compensation. Trophic function failure of placenta changes its hormonal activity, formation of respiratory disorders and development of oxidative stress. The histological structure of placentas among patients with type 1 diabetes mellitus (T1D) is represented by reduced chorionic villi dimensions of all levels. Stromal edema and increased number of mesenchymal stromal cells are found in the stem and intermediate villi, and hypervascularisation and thickening of syncytiocapillary membranes are detected in terminal villi. In type 2 diabetes mellitus (T2D), the histological structure of the placenta may be represented as a premature maturation and abnormal immaturity of the villous tree with focal fibrosis of villi stroma, hypervascularisation of villi, abundance of syncytial nodules and infarction in the subchorial space. The peculiarity of the placenta structure in gestational diabetes mellitus is predominantly an intermediate immature type of development with angiogenesis abnormality. Angiogenesis processes failure and endothelial dysfunction in chorionic villi associated with hyperglycaemia change the permeability of cell membranes, transferring cells to anaerobic respiration. Metabolic imbalance in the placenta causes the development of diabetic micro-angiopathy in the fetal-placental complex, antenatal hypoxia and negative perinatal outcomes.

204-LB: The In Vitro Characteristics of Human Islet Cells from Diverse Donors, Coaggregated with Human Mesenchymal Stromal Cells to form “Neo-islets”, Are Consistently Identical: Relevance to Clinical Trials in Diabetic Subjects

We reported that allogeneic “Neo-Islets” (NIs), organoids composed of ~ equal numbers of Mesenchymal Stromal Cells and culture expanded Islet Cells (ICs) do, when given i.p., omentally engraft and permanently restore euglycemia in auto-immune diabetic NOD mice, i.e., without the use of either encapsulation devices or antirejection drugs. Similarly, spontaneously diabetic pet dogs treated with allogeneic canine NIs (cNIs) significantly improve glycemia with ~ 50% reduction in insulin needs (&amp;gt; 2 years), and this without immune response. In preparation for a Phase I Clinical Trial we tested whether human ICs in NIs (hNIs) are biologically comparable to canine Islet Cells (cICs) and cNIs, i.e., cells from a larger diabetes model. In our preclinical mouse and dog studies, insulin and other islet hormone gene expression levels were used as indicators of ICs in NIs to adequately re-differentiate into insulin and islet-specific endocrine cells when implanted i.p., thereby resulting in physiological control of T1DM.Human islet cells from 6 donors of different demographics were cultured, passaged to P4, and their growth rates, insulin and other islet-associated endocrine genes’ expression levels, secretion of insulin in response to glucose stimulation (GSIS), and NI formation abilities were compared. Results: (1) hICs and hNIs show comparable gene expression profiles to those of cICs and cNIs, and hNIs show identical GSIS responses to those of cNIs. (2) Insulin gene expression profiles of hICs are stable as a function of population doublings and independent of donor diversity. In conclusion, these data indicate that donor variability does not appear to be a significant factor in this NI technology, facilitating our IND work and further demonstrating hNIs’ translational potential in a planned Clinical Trial in subjects with T1DM. Disclosure A. Gooch: Employee; Self; SymbioCellTech. S.S. Chowdhury: Employee; Self; SymbioCellTech. P. Zhang: Employee; Self; SymbioCellTech. Z. Hu: Employee; Self; SymbioCellTech. C. Westenfelder: Consultant; Self; SymbioCellTech.

Using the Quantum Cell Expansion System to Generate Mesenchymal Stromal Cells for Neuroprotection After Cardiopulmonary Bypass

Background & Aim Cardiopulmonary bypass (CPB) has been known to cause substantial systemic inflammation and trigger prolonged microglial activation in the brain. Mesenchymal stromal cells (MSCs) have significant immunomodulatory properties; and we hypothesize that MSCs would be neuroprotective for newborn babies after CPB. However, the number of MSCs required in the pre-clinical and clinical models is in excess of 20 billion. Therefore we tested whether the Quantum could generate sufficient cells for preclinical piglet models and whether the cells were safe and efficacious in vivo. Methods, Results & Conclusion Passage 5-6 MSCs were manufactured in the Quantum using a D5 media containing platelet lysate; and phenotype, trilineage potential, viability, and cell yield were assessed for each lot. To test for safety and efficacy, two-week old piglets (n=16 total) were randomly assigned to one of 3 groups: (1) Control, (2) Cardiopulmonary bypass (CPB), (3) CPB followed by MSC administration. In group 3, MSCs (10 × 10^6 per kg) were delivered through CPB during rewarming period. The brains were fixed three hours after CPB for immunohistochemistry. Microglia was identified with specific antibody to Iba1 in both cortex and white matter. Caspase activation was also evaluated in the cortex. Starting from a mean seeding amount of 17.1 × 10^6 (range: 12.5-20 × 10^6), D5-Platelet lysate media manufactured a mean 4.70 × 10^8 MSCS (range: 3.3-7.5 × 10^8) between 10-12 days at passage 4 or 5 using the Terumo Research-grade expansion set (1.7 sq meters of surface area). The mean viability of MSCs was 92.5% (range: 89.5-92.7%) and all MSCs had >95% CD73, CD90, and CD105 expression and demonstrated trilineage potential. When transferred into the piglet model, immunohistochemistry revealed that MSC treatment normalized microglia expansion and activation caused by CPB to the same levels as the no CPB control. Acute caspase activations in the cortex after CPB were also mitigated by MSC treatment. In conclusion, MSC delivery was safe in an in vivo piglet model during CPB and has the potential to reduce microglial expansion and caspase activation in children undergoing CPB. Further investigation is necessary to determine the effect of MSC delivery through CPB during pediatric cardiac surgery.

Automated Large-Scale Production of Paclitaxel Loaded Mesenchymal Stromal Cells for Cell Therapy Applications.

Mesenchymal stromal cells (MSCs) prepared as advanced therapies medicinal products (ATMPs) have been widely used for the treatment of different diseases. The latest developments concern the possibility to use MSCs as carrier of molecules, including chemotherapeutic drugs. Taking advantage of their intrinsic homing feature, MSCs may improve drugs localization in the disease area. However, for cell therapy applications, a significant number of MSCs loaded with the drug is required. We here investigate the possibility to produce a large amount of Good Manufacturing Practice (GMP)-compliant MSCs loaded with the chemotherapeutic drug Paclitaxel (MSCs-PTX), using a closed bioreactor system. Cells were obtained starting from 13 adipose tissue lipoaspirates. All samples were characterized in terms of number/viability, morphology, growth kinetics, and immunophenotype. The ability of MSCs to internalize PTX as well as the antiproliferative activity of the MSCs-PTX in vitro was also assessed. The results demonstrate that our approach allows a large scale expansion of cells within a week; the MSCs-PTX, despite a different morphology from MSCs, displayed the typical features of MSCs in terms of viability, adhesion capacity, and phenotype. In addition, MSCs showed the ability to internalize PTX and finally to kill cancer cells, inhibiting the proliferation of tumor lines in vitro. In summary our results demonstrate for the first time that it is possible to obtain, in a short time, large amounts of MSCs loaded with PTX to be used in clinical trials for the treatment of patients with oncological diseases.

Effects and long-term follow-up of using umbilical cord blood-derived mesenchymal stromal cells in pediatric patients with severe BK virus-associated late-onset hemorrhagic cystitis after unrelated cord blood transplantation.

This is a retrospective study to evaluate the efficacy and safety of umbilical cord blood-derived mesenchymal stromal cells (MSCs) for the treatment of pediatric patients with severe BK virus-associated late-onset hemorrhagic cystitis (BKV-HC) after unrelated cord blood transplantation (UCBT). Thirteen pediatric patients with severe BKV-HC from December 2013 to December 2015 were treated with MSCs. The number of MSCs transfused in each session was 1×106 /kg once a week until the symptoms improved. The median follow-up time was 1432 (89-2080) days. The median frequency of MSC infusion was 2 (1-3), with eight cured cases and five effective cases; the total efficacy rate was 100%. The copy number of urine BKV DNA was 4.43 (0.36-56.9) ×108 /mL before MSC infusion and 2.67 (0-56.3) ×108 /mL after MSC infusion; the difference was not significant (P=.219). There were no significant differences in the overall survival, disease-free survival, and the incidence of relapse and acute and chronic graft-versus-host disease between the MSC infusion group and non-MSC infusion group. There was also no significant difference in the cytomegalovirus, Epstein-Barr virus (EBV), and fungal and bacterial infection rates between the two groups. Although umbilical cord blood-derived MSCs do not reduce the number of BKV DNA copies in the urine, the cells have a high efficacy rate and minimal side effects in treating severe BKV-HC after UCBT among pediatric patients. MSCs do not affect the rates of relapse, long-term infection, or survival of patients with leukemia.

First Report on Ex Vivo Delivery of Paracrine Active Human Mesenchymal Stromal Cells to Liver Grafts During Machine Perfusion.

Machine perfusion is rapidly becoming the standard for graft preservation and provides new opportunities for organ salvage, reconditioning, and repair. Based on their immune-modulatory and regenerating properties,1 arguable mesenchymal stromal cells (MSCs) are the most promising cell therapy for graft repair on the pump, though proof of concept is still lacking.2 Here, we show the feasibility of delivering clinically relevant numbers of human MSCs (hMSCs; 5–10 × 106/kg) during 30-minute hypothermic oxygenated machine perfusion in porcine liver grafts (n = 8; Figure 1). Full methods are described in Supplemental Materials and Methods (SDC, https://links.lww.com/TP/B821). To track the biodistribution of hMSC, cells were genetically labeled with click beetle red luciferase.3 As shown in Figure 2, bioluminescent imaging of both the anterior and posterior side of the liver showed a wide range and patchy distribution of hMSC which retain throughout the liver after 30 minutes of perfusion. No significant difference between arterial (n = 4) or venous infusion (n = 4) was observed. Histological and RNA-expression analysis confirmed the delivery of the hMSC throughout the liver grafts. About 104–105 hMSC were estimated to be still present per kilogram of liver graft. Importantly, the hMSC retained their paracrine activity after infusion. Using both Luminex and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, increasing levels of human-specific interleukin-6 and interleukin-8 released in porcine blood were shown during 4-hour normothermic perfusion. In conclusion, this is the first report showing an effective delivery of hMSC in a liver machine perfusion model. Evidence of paracrine activity of hMSC after delivery indicates regenerative and immune-modulatory effects during normothermic graft perfusion, though this needs further research in a transplantation model.FIGURE 1.: Graphical abstract of the procedure. The timeline indicates the details of each time point. Pig livers are procured after a warm ischemia period of 30 min and infused with 5 × 106–107 click beetle red luciferase (CBred2)-labeled human mesenchymal stromal cell (hMSC) either via the portal vein or hepatic artery. The distribution of hMSC is visualized by bioluminescence imaging (BLI) immediately (time point 0) after infusion using an IVIS Imaging System 200. The liver is then placed on a purpose-made perfusion device in which it is perfused with cold (10°C) oxygenated University of Wisconsin (UW)-organ preservation fluid. After 30 min of perfusion, the distribution of the hMSC is again imaged using BLI. After imaging, the livers were perfused for 4 h with their own, heparinized, full blood at normothermic temperature (37°C) by ex vivo machine perfusion after which biopsies were collected for further analysis. The BLI signal is quantified, presence of hMSC was assessed in biopsies taken from left and right periphery and center of the liver using human-specific antibodies, proteomic and genetic analysis. HOPE, hypothermic oxygenated machine perfusion; qPCR, quantitative polymerase chain reaction.FIGURE 2.: Effective delivery of paracrine active human mesenchymal stromal cells (hMSCs) during normothermic hepatic machine perfusion. Live imaging of the livers (n = 8) infused with click beetle red luciferase (CBred2)-transduced hMSC in the hepatic artery (n = 4; A, B, E, F) or portal vein (n = 4; C, D, G, H) using the IVIS Imaging System 200, directly after infusion (directly after infusion, A, B, C, D) and after 30 min of perfusion (E, F, G, H). Images are representative images showing bright luciferin-based luminescence at the posterior (A, C, E, G) and anterior side (B, D, F, H) of the liver, demonstrating whole organ coverage of the infused hMSC. The presence of hMSC was confirmed by immunohistochemical staining for copepod green fluorescent protein (copGFP) (I arrows; ×40 objective) at end of perfusion, whereas no staining was observed in liver biopsies before infusion (J). Representative pictures are shown. Human-specific B2M gene expression was determined by nested quantitative polymerase chain reaction (qPCR) to confirm the presence of hMSC in the pig liver at the genetic level (M). Shown are the raw cycle threshold (Ct) values of B2M expression of 3 pigs. B2M expression was measured in the pig samples that were perfused with hMSC, (pig 1–3) and in nonperfused pig livers (n = 3). Luminex-based analysis using human-specific cytokines showed that interleukin (IL)-6 (O) and IL-8 (P) was secreted in the liver perfusate in 2 of the 4 pigs that were infused via the hepatic artery (filled symbols). In pig livers that were not infused with hMSC, no IL-6 or IL-8 was detected (open symbols). Human-specific IL-6 and IL-8 protein release was confirmed in the same 2 pig livers by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (data not shown). nd, not detectable.

Endothelial Progenitor and Mesenchymal Stromal Cells in Newborns With Congenital Diaphragmatic Hernia Undergoing Extracorporeal Membrane Oxygenation.

Background: Endothelial progenitor (EPC) and mesenchymal stromal cells (MSC) can regenerate damaged endothelium and thereby improve pulmonary endothelial dysfunction. We do not know, how extracorporeal membrane oxygenation (ECMO) might affect EPC- and MSC-mediated regenerative pathways in patients with congenital diaphragmatic hernia (CDH). Therefore, we investigated, if ECMO support impacts EPC and MSC numbers in CDH patients.Methods: Peripheral blood mononuclear cells from newborns with ECMO-dependent (n = 18) and ECMO-independent CDH (n = 12) and from healthy controls (n = 12) were isolated. The numbers of EPC and MSC were identified by flowcytometry. Serum levels of vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-2 were determined.Results: EPC and MSC were elevated in newborns with CDH. ECMO-dependent infants had higher EPC subpopulation counts (2,1–7,6-fold) before treatment compared to ECMO-independent infants. In the disease course, EPC and MSC subpopulation counts in ECMO-dependent infants were lower than before ECMO initiation. During ECMO, VEGF serum levels were significantly reduced (by 90.5%) and Ang2 levels significantly increased (by 74.8%).Conclusions: Our data suggest that ECMO might be associated with a rather impaired mobilization of EPC and MSC and with a depression of VEGF serum levels in newborns with CDH.