The CIt protocol: A blueprint to potentiate the immunogenicity of immunoproteasome-reprogrammed mesenchymal stromal cells.

Immunoproteasome-reprogrammed mesenchymal stromal cells (IRMs) can surpass dendritic cells at eliciting tumor-specific immunity. However, the current IRM vaccination regimen remains clinically unsuitable due to the relatively high dose of IRMs needed. Since the administration of a lower IRM dose triggers a feeble anti-tumoral response, we aimed to combine this vaccination regimen with different modalities to fine-tune the potency of the vaccine. In a nutshell, we found that the co-administration of IRMs and interleukin-12 accentuates the anti-tumoral response, whereas the cross-presentation potency of IRMs is enhanced via intracellular succinate build-up, delayed endosomal maturation, and increased endosome-to-cytosol plasticity. Stimulating phagocyte-mediated cancer efferocytosis by blocking the CD47-SIRPα axis was also found to enhance IRM vaccine outcomes. Upon designing a single protocol combining the abovementioned strategies, 60% of treated animals exhibited a complete response. Altogether, this is the first IRM-based vaccination study, optimized to simultaneously target three vaccine-related pitfalls: T-cell response, antigen cross-presentation, and cancer phagocytosis.

UM171A-induced ROS promote antigen cross-presentation of immunogenic peptides by bone marrow-derived mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cells (MSCs) have been extensively used in the clinic due to their exquisite tissue repair capacity. However, they also hold promise in the field of cellular vaccination as they can behave as conditional antigen presenting cells in response to interferon (IFN)-gamma treatment under a specific treatment regimen. This suggests that the immune function of MSCs can be pharmacologically modulated. Given the capacity of the agonist pyrimido-indole derivative UM171a to trigger the expression of various antigen presentation-related genes in human hematopoietic progenitor cells, we explored the potential use of UM171a as a means to pharmacologically instill and/or promote antigen presentation by MSCs. METHODS: Besides completing a series of flow-cytometry-based phenotypic analyses, several functional antigen presentation assays were conducted using the SIINFEKL-specific T-cell clone B3Z. Anti-oxidants and electron transport chain inhibitors were also used to decipher UM171a's mode of action in MSCs. Finally, the potency of UM171a-treated MSCs was evaluated in the context of therapeutic vaccination using immunocompetent C57BL/6 mice with pre-established syngeneic EG.7T-cell lymphoma. RESULTS: Treatment of MSCs with UM171a triggered potent increase in H2-Kb cell surface levels along with the acquisition of antigen cross-presentation abilities. Mechanistically, such effects occurred in response to UM171a-mediated production of mitochondrial-derived reactive oxygen species as their neutralization using anti-oxidants or Antimycin-A mitigated MSCs' ability to cross-present antigens. Processing and presentation of the immunogenic ovalbumin-derived SIINFEKL peptide was caused by de novo expression of the Psmb8 gene in response to UM171a-triggered oxidative stress. When evaluated for their anti-tumoral properties in the context of therapeutic vaccination, UM171a-treated MSC administration to immunocompetent mice with pre-established T-cell lymphoma controlled tumor growth resulting in 40% survival without the need of additional supportive therapy and/or standard-of-care. CONCLUSIONS: Altogether, our findings reveal a new immune-related function for UM171a and clearly allude to a direct link between UM171a-mediated ROS induction and antigen cross-presentation by MSCs. The fact that UM171a treatment modulates MSCs to become antigen-presenting cells without the use of IFN-gamma opens-up a new line of investigation to search for additional agents capable of converting immune-suppressive MSCs to a cellular tool easily adaptable to vaccination.

Effects of Oxygen and Glucose on Bone Marrow Mesenchymal Stem Cell Culture.

This study determines whether the viability of mesenchymal stem cell (MSC) in vitro is most sensitive to oxygen supply, energetic substrate supply, or accumulation of lactate. Mouse unmodified (wild type (WT)) and erythropoietin (EPO) gene-modified MSC is cultured for 7 days in normoxic (21%) and anoxic conditions. WT-MSC is cultured in anoxia for 45 days in high and regular glucose media and both have similar viability when compared to their normoxic controls at 7 days. Protein production of EPO-MSC is unaffected by the absence of oxygen. MSC doubling time and post-anoxic exposure is increased (WT: 32.3-73.3 h; EPO: 27.2-115 h). High glucose leads to a 37% increase in cell viability at 13 days and 17% at 30 days, indicating that MSC anoxic survival is affected by supply of metabolic substrate. However, after 30 days, little difference in viability is found, and at 45 days, complete cell death occurs in both the conditions. This death cannot be attributed to lack of glucose or lactate levels. MSC stemness is retained for both osteogenic and adipogenic differentiations. The absence of oxygen increases the doubling time of MSC but does not affect their viability, protein production, or differentiation capacity.

Endothelial nitric oxide synthase overexpressing human early outgrowth cells inhibit coronary artery smooth muscle cell migration through paracrine functions.

Cells mobilized from the bone marrow can contribute to endothelial regeneration and repair. Nevertheless, cardiovascular diseases are associated with diminished numbers and function of these cells, attenuating their healing potential. Gene transfer of endothelial nitric oxide synthase (eNOS) can restore the activity of circulating cells. Furthermore, estrogen accelerates the reendothelialization capacity of early outgrowth cells (EOCs). We hypothesized that overexpressing eNOS alone or in combination with estrogen stimulation in EOCs would potentiate the beneficial effects of these cells in regulating smooth muscle cell (SMC) function. Native human EOCs did not have any effect on human coronary artery SMC (hCASMC) proliferation or migration. Transfecting EOCs with a human eNOS plasmid and/or stimulating with 17ß-estradiol (E2) increased NO production 3-fold and enhanced EOC survival. Moreover, in co-culture studies, eNOS overexpressing or E2-stimulated EOCs reduced hCASMC migration (by 23% and 56% respectively), vs. control EOCs. These effects do not implicate ERK1/2 or focal adhesion kinases. Nevertheless, NOS-EOCs had no effect on hCASMC proliferation. These results suggest that overexpressing or activating eNOS in EOCs increases their survival and enhances their capacity to regulate SMC migration through paracrine effects. These data elucidate how eNOS overexpression or activation in EOCs can prevent vascular remodeling.

Radiation-Induced Oral Mucositis.

Radiation-induced oral mucositis (RIOM) is a major dose-limiting toxicity in head and neck cancer patients. It is a normal tissue injury caused by radiation/radiotherapy (RT), which has marked adverse effects on patient quality of life and cancer therapy continuity. It is a challenge for radiation oncologists since it leads to cancer therapy interruption, poor local tumor control, and changes in dose fractionation. RIOM occurs in 100% of altered fractionation radiotherapy head and neck cancer patients. In the United Sates, its economic cost was estimated to reach 17,000.00 USD per patient with head and neck cancers. This review will discuss RIOM definition, epidemiology, impact and side effects, pathogenesis, scoring scales, diagnosis, differential diagnosis, prevention, and treatment.

Adipose mesenchymal stromal cells minimize and repair radiation-induced oral mucositis.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have been used to minimize and repair radiation-induced normal tissue injury in the intestine, salivary gland, liver, skin, lungs and cardiac muscle. This study investigated the ability of adipose tissue-derived MSCs (aMSCs) to minimize and/or repair single dose radiation-induced oral mucositis (RIOM). METHODS: Syngenic phenotypically and functionally characterized BALB/c mouse aMSCs were implanted intraperitoneally in a RIOM mouse model with different dosing protocols. Response was quantified macroscopically, microscopically and by using different histological and clinically relevant parameters. RESULTS: Irradiation at 18 Gy generated a self-resolved single-dose RIOM BALB/c mouse model with 5.6 ± 0.3 days mean duration (95% confidence interval (CI) 4.233-7.1 days) and 100% survival rate. Intraperitoneal implantation of 5 doses of 2.5 million freshly cultured syngenic aMSCs significantly and reproducibly reduced RIOM ulcer duration to 1.6 ± 0.3 days (95% CI 0.0233-3.1 days, a 72% reduction in RIOM ulcer duration), ulcer size and ulcer floor epithelial height. The therapeutic benefits were significantly dependent on dose size and frequency, number of doses, and therapy onset time. aMSCs therapy significantly minimized the RIOM-related weight loss, accelerated the weight gain and improved irradiated animals' hydration and nutritional status. aMSCs therapy did not potentiate head and neck cancer in vitro. CONCLUSIONS: Syngenic freshly cultured aMSCs significantly minimized and repaired radiation-induced oral mucositis with a 72% reduction in ulcer duration. aMSCs dose size and frequency, number of doses and therapy onset time are the main keys for optimized therapeutic outcome. aMSCs therapy did not stimulate Head and Neck cancer cell growth in-vitro.

Single-Dose Radiation-Induced Oral Mucositis Mouse Model.

The generation of a self-resolved radiation-induced oral mucositis (RIOM) mouse model using the highest possibly tolerable single ionizing radiation (RT) dose was needed in order to study RIOM management solutions. We used 10-week-old male BALB/c mice with average weight of 23 g for model production. Mice were treated with an orthovoltage X-ray irradiator to induce the RIOM ulceration at the intermolar eminence of the animal tongue. General anesthesia was injected intraperitoneally for proper animal immobilization during the procedure. Ten days after irradiation, a single RT dose of 10, 15, 18, 20, and 25 Gy generated a RIOM ulcer at the intermolar eminence (posterior upper tongue surface) with mean ulcer floor (posterior epithelium) heights of 190, 150, 25, 10, and 10 µm, respectively, compared to 200 µm in non-irradiated animals. The mean RIOM ulcer size % of the total epithelialized upper surface of the animal tongue was RT dose dependent. At day 10, the ulcer size % was 2, 5, 27, and 31% for 15, 18, 20, and 25 Gy RT, respectively. The mean relative surface area of the total epithelialized upper surface of the tongue was RT dose dependent, since it was significantly decreased to 97, 95, 88, and 38% with 15, 18, 20, and 25 Gy doses, respectively, at day 10 after RT. Subcutaneous injection of 1 mL of 0.9% saline/6 h for 24 h yielded a 100% survival only with 18 Gy self-resolved RIOM, which had 5.6 ± 0.3 days ulcer duration. In conclusion, we have generated a 100% survival self-resolved single-dose RIOM male mouse model with long enough duration for application in RIOM management research. Oral mucositis ulceration was radiation dose dependent. Sufficient hydration of animals after radiation exposure significantly improved their survival.

Adipose mesenchymal stromal cells response to ionizing radiation.

BACKGROUND AIMS: This study evaluates the biological response of adipose tissue-derived mesenchymal stromal cells (aMSCs) to ionizing radiation (IR). METHODS: Irradiated BALB/c mice aMSCs were characterized for functionality and phenotype. The clonogenic capacity of irradiated aMSCs was assessed and compared with those of metastatic breast cancer cell line (4T1) and normal mouse fibroblasts (NIH3T3-wt). We investigated the IR-induced DNA damage response, apoptosis, changes in cell cycle (CC) dynamics and protein and gene expression. RESULTS: Irradiated and non-irradiated aMSCs were able to differentiate into adipocytes, chondrocytes and osteocytes with no significant difference. Irradiated aMSCs maintained the expression of mesenchymal stromal cells (MSCs) surface antigens and, as expected, were negative for hematopoietic stem cells (HSCs) surface antigens when tested up to 7 days after IR for all irradiation doses with no significant difference. Clonogenically, irradiated aMSCs had higher relative survival fraction and plating efficiency than 4T1 and NIH3T3-wt. Irradiated aMSCs expressed higher □H2AX and significantly showed faster and more time-efficient IR-induced DNA damage response evident by up-regulated DNA-PKcs and RAD51. Two hours after IR, most of aMSCs DNA damage/repair-related genes showed up-regulation that disappeared within 6 h after IR. Irradiated aMSCs showed a significant rise and an earlier peak of p-ATM-dependent and -independent (p84/5E10-mediated) G2/M CC arrest compared with 4T1 and NIH3T3-wt. CONCLUSIONS: After IR exposure, aMSCs showed a robust and time-efficient radiation-induced DNA damage repair response, stable phenotypical characteristics and multi-lineage differentiation potential, suggesting they may be reliable candidates for cell therapy in radiation oncology regenerative medicine.

Melatonin pretreatment of human adipose tissue-derived mesenchymal stromal cells enhances their prosurvival and protective effects on human kidney cells.

The efficacy of cell therapy for many diseases can be limited by the poor survival of implanted cells in an environment of tissue injury. Melatonin has been reported to have antioxidative and antiapoptotic effects. Adipose tissue-derived mesenchymal stromal cells (ASCs), cells easily obtained in high amounts and with minimal discomfort, have shown great promise in cell therapy applications, such as in acute kidney injury. We hypothesized that melatonin pretreatment of human ASCs (hASCs) would improve their renoprotective and prosurvival effects. We therefore investigated the action of melatonin on hASCs, as well as the effect of the resulting hASCs-conditioned media (CM) on human kidney cells exposed to oxidative and apoptotic injury-provoking doses of cisplatin. Our results demonstrated that pretreatment of hASCs with melatonin, 100 µM for 3 h, significantly increased their proliferation and their expression of prosurvival P-Erk1/2 and P-Akt, and of antioxidative enzymes catalase and heme oxygenase (HO)-1. In addition, the CM from hASCs pretreated with melatonin provoked a significantly higher proliferation and migration of HK-2 human kidney epithelial cells. Furthermore, this CM exerted significantly higher prosurvival and antiapoptotic actions on HK-2 cells exposed to cisplatin in vitro. Western blot analysis showed higher expression of P-Erk1/2, Bcl-2, SOD-1, and HO-1 in the HK-2 cells exposed to cisplatin in the presence of CM from melatonin-pretreated hASCs. In sum, our study revealed that in vitro pretreatment of hASCs with melatonin may significantly enhance their survival and their therapeutic effectiveness on injured tissue.

Human mesenchymal stromal cells transiently increase cytokine production by activated T cells before suppressing T-cell proliferation: effect of interferon-γ and tumor necrosis factor-α stimulation.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) suppress T-cell proliferation, especially after activation with inflammatory cytokines. We compared the dynamic action of unprimed and interferon (IFN)-γ plus tumor necrosis factor (TNF)-α-pretreated human bone marrow-derived MSCs on resting or activated T cells. METHODS: MSCs were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) at high MSC-to-PBMC ratios in the absence or presence of concomitant CD3/CD28-induced T-cell activation. The kinetic effects of MSCs on cytokine production and T-cell proliferation, cell cycle and apoptosis were assessed. RESULTS: Unprimed MSCs increased the early production of IFN-γ and interleukin (IL)-2 by CD3/CD28-activated PBMCs before suppressing T-cell proliferation. In non-activated PBMC co-cultures, low levels of IL-2 and IL-10 synthesis were observed with MSCs in addition to low levels of CD69 expression by T cells and no T-cell proliferation. MSCs also decreased apoptosis in resting and activated T cells and inhibited the transition of these cells into the sub-G0/G1 and the S phases. With inhibition of indoleamine 2,3 dioxygenase, MSCs increased CD3/CD28-induced T-cell proliferation. After priming with IFN-γ plus TNF-α, MSCs were less potent at increasing cytokine production by CD3/CD28-activated PBMCs and more effective at inhibiting T-cell proliferation but had preserved anti-apoptotic functions. CONCLUSIONS: Unprimed MSCs induce a transient increase in IFN-γ and IL-2 synthesis by activated T cells. Pre-treatment of MSCs with IFN-γ plus TNF-α may increase their effectiveness and safety in vivo.

Stem cell microvesicles transfer cystinosin to human cystinotic cells and reduce cystine accumulation in vitro.

Cystinosis is a rare disease caused by homozygous mutations of the CTNS gene, encoding a cystine efflux channel in the lysosomal membrane. In Ctns knockout mice, the pathologic intralysosomal accumulation of cystine that drives progressive organ damage can be reversed by infusion of wildtype bone marrow-derived stem cells, but the mechanism involved is unclear since the exogeneous stem cells are rarely integrated into renal tubules. Here we show that human mesenchymal stem cells, from amniotic fluid or bone marrow, reduce pathologic cystine accumulation in co-cultured CTNS mutant fibroblasts or proximal tubular cells from cystinosis patients. This paracrine effect is associated with release into the culture medium of stem cell microvesicles (100-400 nm diameter) containing wildtype cystinosin protein and CTNS mRNA. Isolated stem cell microvesicles reduce target cell cystine accumulation in a dose-dependent, Annexin V-sensitive manner. Microvesicles from stem cells expressing CTNS(Red) transfer tagged CTNS protein to the lysosome/endosome compartment of cystinotic fibroblasts. Our observations suggest that exogenous stem cells may reprogram the biology of mutant tissues by direct microvesicle transfer of membrane-associated wildtype molecules.

Erythropoietin gene-enhanced marrow mesenchymal stromal cells decrease cisplatin-induced kidney injury and improve survival of allogeneic mice.

Bone marrow-derived mesenchymal stromal cells (MSCs) are promising for regenerative medicine applications, such as for renoprotection and repair in acute kidney injury (AKI). Erythropoietin (Epo) can also exert cytoprotective effects on various tissues including the kidney. We hypothesized that MSCs gene-enhanced to secrete Epo may produce a significant beneficial effect in AKI. Mouse Epo-secreting MSCs were generated, tested in vitro, and then implanted by intraperitoneal injection in allogeneic mice previously administered cisplatin to induce AKI. Epo-MSCs significantly improved survival of implanted mice as compared to controls (67% survival versus 33% with Vehicle only). Also, Epo-MSCs led to significantly better kidney function as shown by lower levels of blood urea nitrogen (72 ± 9.5 mg/dl versus 131 ± 9.20 mg/dl) and creatinine (74 ± 17 µmol/l versus 148±19.4 µmol/l). Recipient mice also showed significantly decreased amylase and alanine aminotransferase blood concentrations. Kidney sections revealed significantly less apoptotic cells and more proliferating cells. Furthermore, PCR revealed the presence of implanted cells in recipient kidneys, with Epo-MSCs leading to significantly increased expression of Epo and of phosphorylated-Akt (Ser473) (P-Akt) in these kidneys. In conclusion, our study demonstrates that Epo gene-enhanced MSCs exert significant tissue protective effects in allogeneic mice with AKI, and supports the potential use of gene-enhanced cells as universal donors in acute injury.

A variant OSR1 allele which disturbs OSR1 mRNA expression in renal progenitor cells is associated with reduction of newborn kidney size and function.

Human nephrons are formed during fetal life through an interaction between the branching ureteric bud and progenitor cells. The wide variation in final nephron number has been attributed to allelic variants of genes regulating ureteric bud arborization. Here, we hypothesize that dysfunctional variants of the Odd-Skipped Related 1 (OSR1) gene which compromise the renal progenitor cell pool might also limit newborn kidney size and function. We show that OSR1 is expressed in human mesenchymal stem cells, the blastemal component of Wilms tumors and CD24+/CD133+ progenitor cells isolated from the mature kidney. We identified an OSR1(rs12329305(T)) allele in 6% of normal Caucasians which alters an exon2 splice enhancer. This variant is predicted to reduce spliceosome-binding affinity and stability of the OSR1 mRNA. In cultured cells, the OSR1(rs12329305)(T) allele produced no identifiable transcript. Normal Caucasian newborns from Montreal with the OSR1(rs12329305)(T) allele had kidney volume 11.8% smaller (P= 0.006) and cord blood cystatin C levels 12.6% higher (P = 0.005) than those with wild-type genotype. Effects of the OSR1(rs12329305)(T) allele are additive with genes that alter ureteric bud branching. Kidney volume was reduced more in newborns bearing both RET(rs1800860)(A) and OSR1(rs12329305)(T) alleles (22%, P= 0.0008) and cystatin C was increased by 17% (P= 0.006) versus newborns with wild-type alleles. Although only two subjects had PAX2(rs11599825)(A) and OSR1(rs12329305)(T) alleles, kidney size was reduced by 27% and cystatin C was increased by 14% versus wild-types (P= NS).

Bone marrow mesenchymal stromal cell therapy for external urethral sphincter restoration in a rat model of stress urinary incontinence.

OBJECTIVE: To assess the effect of intra-sphincteric injections of bone marrow mesenchymal stromal cells (MSCs) on Valsalva leak point pressure (VLPP) changes in an animal model of stress urinary incontinence (SUI). MATERIALS AND METHODS: Twenty-four female Sprague-Dawley rats underwent bilateral pudendal nerve section to induce SUI. Six rats were SUI controls, 6 received periurethral injection of Plasma-Lyte (SUI placebo control) and 12 were given periurethral injection of PKH26-labeled MSCs. Four weeks after injection, conscious cystometry was undertaken in animals and VLPP recorded. All groups were sacrificed, and frozen urethra sections were submitted to pathology and immunohistochemistry assessment. RESULTS: Rat MSCs were positive for the cell surface antigens CD44, CD73, CD90, and RT1A, and negative for CD31, CD45, and RT1B, confirming their stem cell phenotype. In vitro, differentiated MSCs expressed α-smooth muscle actin (SMA) and desmin, markers of smooth and striated muscles in vivo. Immunohistochemistry of rat urethras revealed PKH26-labeled MSCs in situ and at the injection site. LPP was significantly improved in animals injected with MSCs. Mean LPP was 24.28 ± 1.47 cmH(2) O in rats implanted with MSCs and 16.21 ± 1.26 cmH(2) O in SUI controls (P<0.001). Atrophic urethras with implanted MSCs were positively stained for myosin heavy chain and desmin. CONCLUSION: Rat MSCs have the ability to differentiate and skew their phenotype towards smooth and striated muscles, as demonstrated by SMA up-regulation and desmin expression. Periurethral injection of MSCs in an animal model of SUI restored the damaged external urethral sphincter and significantly improved VLPP.

Human marrow-derived mesenchymal stromal cells decrease cisplatin renotoxicity in vitro and in vivo and enhance survival of mice post-intraperitoneal injection.

Acute kidney injury (AKI) can occur from the toxic side-effects of chemotherapeutic agents such as cisplatin. Bone marrow-derived mesenchymal stromal cells (MSCs) have demonstrated wide therapeutic potential often due to beneficial factors they secrete. The goal of this investigation was to evaluate in vitro the effect of human MSCs (hMSCs) secretome on cisplatin-treated human kidney cells, and in vivo the consequence of hMSCs intraperitoneal (ip) implantation in mice with AKI. Our results revealed that hMSCs-conditioned media improved survival of HK-2 human proximal tubular cells exposed to cisplatin in vitro. This enhanced survival was linked to increased expression of phosphorylated Akt (Ser473) and was reduced by a VEGF-neutralizing antibody. In vivo testing of these hMSCs established that ip administration in NOD-SCID mice decreased cisplatin-induced kidney function impairment, as demonstrated by lower blood urea nitrogen levels and higher survival. In addition, blood phosphorous and amylase levels were also significantly decreased. Moreover, hMSCs reduced the plasma levels of several inflammatory cytokines/chemokines. Immunohistochemical examination of kidneys showed less apoptotic and more proliferating cells. Furthermore, PCR indicated the presence of hMSCs in mouse kidneys, which also showed enhanced expression of phosphorylated Akt. In conclusion, our study reveals that hMSCs can exert prosurvival effects on renal cells in vitro and in vivo, suggests a paracrine contribution for kidney protective abilities of hMSCs delivered ip, and supports their clinical potential in AKI.

Targeted gene addition to human mesenchymal stromal cells as a cell-based plasma-soluble protein delivery platform.

BACKGROUND AIMS: Gene-modified mesenchymal stromal cells (MSC) provide a promising tool for cell and gene therapy-based applications by potentially acting as a cellular vehicle for protein-replacement therapy. However, to avoid the risk of insertional mutagenesis, targeted integration of a transgene into a 'safe harbor' locus is of great interest. METHODS: We sought to determine whether zinc finger nuclease (ZFN)-mediated targeted addition of the erythropoietin (Epo) gene into the chemokine [C-C motif] receptor 5 (CCR5) gene locus, a putative safe harbor locus, in MSC would result in stable transgene expression in vivo. RESULTS: Whether derived from bone marrow (BM), umbilical cord blood (UCB) or adipose tissue (AT), 30-40% of human MSC underwent ZFN-driven targeted gene addition, as determined by a combination of fluorescence-activated cell sorting (FACS)- and polymerase chain reaction (PCR)-based analyzes. An enzyme-linked immunosorbent assay (ELISA)-based analysis of gene-targeted MSC expressing Epo from the CCR5 locus showed that these modified MSC were found to secrete a significant level of Epo (c. 2 IU/10(6)cells/24 h). NOD/SCID/gammaC mice injected with ZFN-modified MSC expressing Epo exhibited significantly higher hematocrit and Epo plasma levels for several weeks post-injection, compared with mice receiving control MSC. CONCLUSIONS: These data demonstrate that MSC modified by ZFN-driven targeted gene addition may represent a cellular vehicle for delivery of plasma-soluble therapeutic factors.

Neo-organoid of marrow mesenchymal stromal cells secreting interleukin-12 for breast cancer therapy.

Bone marrow-derived mesenchymal stromal cells (MSCs), beneficial for regenerative medicine applications due to their wide differentiation capabilities, also hold promise as cellular vehicles for the delivery of therapeutic plasma-soluble gene products due to their ease of handling, expansion, and genetic engineering. We hypothesized that MSCs, gene enhanced to express interleukin-12 (IL-12) and then embedded in a matrix, may act as an anticancer neo-organoid when delivered s.c. in autologous/syngeneic hosts. We performed such experiments in mice and noted that primary murine MSCs retrovirally engineered to secrete murine IL-12 can significantly interfere with growth of 4T1 breast cancer cells in vivo, with a more substantial anticancer action achieved when these cells are embedded in a matrix. Plasma of mice that received the IL-12 MSC-containing neo-organoids showed increased levels of IL-12 and IFN-gamma. Histopathologic analysis revealed less tumor cells in implants of 4T1 cells with IL-12 MSCs, and the presence of necrotic tumor islets and necrotic capillaries, suggesting antiangiogenesis. We also showed that the anticancer effect exerted by the IL-12 MSCs is immune mediated because it is absent in immunodeficient mice, is not due to systemic IL-12 delivery, and also occurs in a B16 melanoma model. This study therefore establishes the feasibility of using gene-enhanced MSCs in a cell-based neo-organoid approach for cancer treatment.

Coupling erythropoietin secretion to mesenchymal stromal cells enhances their regenerative properties.

AIMS: Mesenchymal stromal cells (MSCs) possess intrinsic features that identify them as useful for treating ischaemic syndromes. Poor in vivo survival/engraftment of MSCs, however, limits their overall effectiveness. In this work, we tested whether genetically engineering MSCs to secrete erythropoietin (Epo) could represent a better therapeutic platform than MSCs in their native form. METHODS AND RESULTS: MSCs from C57Bl/6 mice were retrovirally transduced with either an empty vector or one that causes the production of Epo and were then analysed for the alterations in angiogenic and survival potential. Using a mouse model of myocardial infarction (MI), the regenerative potential of null MSCs and Epo-overexpressing MSCs (Epo+MSCs) was assessed using serial echocardiogram and invasive haemodynamic measurements. Infarct size, capillary density and neutrophil influx were assessed using histologic techniques. Using in vitro assays coupled with an in vivo Matrigel plug assay, we demonstrate that engineering MSCs to express Epo does not alter their immunophenotype or plasticity. However, relative to mock-modified MSCs [wild-type (WT)-MSCs], Epo+MSCs are more resilient to apoptotic stimuli and initiate a more robust host-derived angiogenic response. We also identify and characterize the autocrine loop established on MSCs by having them secrete Epo. Furthermore, in a murine model of MI, animals receiving intracardiac injections of Epo+MSCs exhibited significantly enhanced cardiac function compared with WT-MSCs and saline-injected control animals post-MI, owing to the increased myocardial capillary density and the reduced neutrophilia. CONCLUSION: Epo overexpression enhances the cellular regenerative properties of MSCs by both autocrine and paracrine pathways.

CD34 expression on murine marrow-derived mesenchymal stromal cells: impact on neovascularization.

OBJECTIVE: CD34 is a sialomucin often expressed by cells with hemangiopoietic potential and widely serves as a surrogate marker of stem cell potential. Mesenchymal stromal cells (MSCs) also express CD34, although the functional significance of its expression remains undefined. In this study, we determined whether CD34(pos) MSCs are functionally distinct from CD34(null) MSCs. MATERIALS AND METHODS: MSCs derived from C57Bl/6 mice were transduced to express the green fluorescent protein (GFP) from which pure CD34(pos) MSC and CD34(null) MSC clones were selected. In vitro, clones were examined by microarray analysis, while in vivo subcutaneous implantation of matrix-embedded MSCs was used to assess cell survival, differentiation, and neovascularization. RESULTS: The flow cytometric phenotype of CD34(pos) and CD34(null) MSCs were similar, as was gene expression of vascular endothelial growth factors (VEGFs) A and B. However, CD34(pos) MSCs upregulated a number of supplementary angiogenesis-associated genes and showed a greater expression of gene associated with vascular differentiation. At 15 days postimplantation, cell survival between CD34(pos) and CD34(null) MSCs was similar, however, CD34(pos) MSCs evoked a significantly greater host-derived response (4.2 +/- 0.7 vs 1.9 +/- 0.5 x 10(6) cells; p < 0.05). GFP-expressing CD34(pos) MSC implants acquired significantly more CD31 expression compared to CD34(null) MSC cells (10.7% +/- 8.4% vs 3.1% +/- 0.6%; p < 0.05), as well as a significantly greater host-derived endothelial cell influx (CD31(+)/CD45(-)). CONCLUSION: CD34 expression by MSCs correlates with enhanced vasculogenic and angiogenic potential in vivo.

Design and fabrication of 3D porous scaffolds to facilitate cell-based gene therapy.

Biomaterials capable of efficient gene delivery by embedded cells provide a fundamental tool for the treatment of acquired or hereditary diseases. A major obstacle is maintaining adequate nutrient and oxygen diffusion to cells within the biomaterial. In this study, we combined the solid free-form fabrication and porogen leaching techniques to fabricate three-dimensional scaffolds, with bimodal pore size distribution, for cell-based gene delivery. The objective of this study was to design micro-/macroporous scaffolds to improve cell viability and drug delivery. Murine bone marrow-derived mesenchymal stromal cells (MSCs) genetically engineered to secrete erythropoietin (EPO) were seeded onto poly-L-lactide (PLLA) scaffolds with different microporosities. Over a period of 2 weeks in culture, an increase in cell proliferation and metabolic activity was observed with increasing scaffold microporosity. The concentration of EPO detected in supernatants also increased with increasing microporosity level. Our study shows that these constructs can promote cell viability and release of therapeutic proteins, and clearly demonstrates their capacity for a dual role as scaffolds for tissue regeneration and as delivery systems for soluble gene products.