Thread- Reinforced Encapsulation Devices (THRED) for Therapeutic Stem Cell Delivery

BackgroundBone mesenchymal stromal cells (BMSC) showed protective potential against intestinal ischemia. Oxygenase-1(HO-1) could alleviate oxidative stress. In the present study, we constructed HO-1-expressing BMSC and detected the effects of it on survival, intestinal injury and inflammation following intestinal ischemia and reperfusion injury (I/R).MethodsIn this experiment, eighty adult male mice were divided into Sham, I/R, I/R + BMSC, I/R + BMSC/HO-1 groups. Mice were anesthetized and intestinal I/R model were established by temporarily occluding the superior mesenteric artery for 60 min with a non-crushing clamp. Following ischemia, the clamp was removed and the intestines were allowed for reperfusion. Prior to abdominal closure, BMSC/ HO-1 (2 × 106 cells) or BMSC (2 × 106 cells) were injected into the peritoneum of I/R mice respectively. Mice were allowed to recover for 24 h and then survival rate, intestinal injury and inflammation were determined. Reactive oxygen species (ROS) was assayed by fluorescent probe. TNFα and IL-6 were assayed by ELISA.ResultsBMSC/HO-1 increased seven day survival rate, improved intestinal injury and down-regulated inflammation after intestinal I/R when compared with sole BMSC (p < 0.05 respectively). Multiple pro-inflammatory media were also decreased following application of BMSC/HO-1, when compared with sole BMSC (p < 0.05) respectively, suggesting that BMSC /HO-1 had a better protection to intestinal I/R than BMSC therapy.ConclusionAdministration of BMSC/HO-1 following intestinal I/R, significantly improved intestinal I/R by limiting intestinal damage and inflammation.

Introduction: Intestinal ischemia remains a frequent pathology with a mortality rate up to 80%, due to delayed diagnosis and lack of efficient therapy. Intestinal reperfusion enhances the catastrophic effect of ischemia, known as ischemia-reperfusion injury (IRI). In contrast to other organs, serological markers for intestinal IRI are missing. We have accumulated evidence that villin-1 -a protein anchoring the actin filaments at the epithelial brush border- is a valuable candidate.The aim was to analyze villin-1 as a serological biomarker in a rodent and human model of intestinal ischemia reperfusion injury (IRI). Methods: In a rat model of intestinal IRI (temporary mesenteric artery clamping), 4 conditions were tested: (i) laparotomy only (sham); (ii) 30min ischemia + 5 reperfusion periods (0min/30min/60min/120min/24hours); (iii) 45min ischemia + 5 reperfusion periods; (iv) 60min ischemia + 5 reperfusion periods; (n=6/group). For survival analysis, 7-day reperfusion was included in each condition (n=10/group). Other end-points that were analyzed: histology (Park-Chiu/villus length); intestinal permeability (Ussing chamber); villin-1 (Western-Blot). In a validated human model of intestinal IRI (6cm jejunal-clamping during pancreaticoduodenectomy; 45min ischemia + 0min/30min/120min reperfusion) villin-1 was analyzed by immunoprecipitation (n=6). Results: In rat, increasing ischemia resulted in decreased survival (30min: 90%; 45min: 50%; 60min: 10%) and loss of intestinal integrity (histology/permeability). From 45min ischemia, villin-1 appeared in the plasma at 0min reperfusion and remained detectable until 120min reperfusion (Figure 1). At 0min reperfusion, villin-1 could differentiate between 45min or 60min ischemia corresponding to the different survival. Overall, villin-1 had a strong correlation with Park-Chiu score (r=0.7954;p<0.0001); villus length (r=−0.6585;p<0.0001); and permeability (r=−0.6127;p=0.0019). In human, villin-1 was released with ischemia and remained detectable until 120min reperfusion (Figure 2). Conclusion: For the first time, we showed that villin-1 is a serological biomarker of intestinal IRI in rat and human. These findings open new perspectives in the diagnosis of intestinal ischemia and warrant further clinical investigations.FIGURE 1: Rat model.FIGURE 2: Human model.

SummaryThe gap in knowledge of the molecular mechanisms underlying differentiation of human pluripotent stem cells (hPSCs) into the mesenchymal cell lineages hinders the application of hPSCs for cell-based therapy. In this study, we identified a critical role of muscle segment homeobox 2 (MSX2) in initiating and accelerating the molecular program that leads to mesenchymal stem/stromal cell (MSC) differentiation from hPSCs. Genetic deletion of MSX2 impairs hPSC differentiation into MSCs. When aided with a cocktail of soluble molecules, MSX2 ectopic expression induces hPSCs to form nearly homogeneous and fully functional MSCs. Mechanistically, MSX2 induces hPSCs to form neural crest cells, an intermediate cell stage preceding MSCs, and further differentiation by regulating TWIST1 and PRAME. Furthermore, we found that MSX2 is also required for hPSC differentiation into MSCs through mesendoderm and trophoblast. Our findings provide novel mechanistic insights into lineage specification of hPSCs to MSCs and effective strategies for applications of stem cells for regenerative medicine.

Survivin Is a Master Regulator of Mesenchymal Stem Cell Functions and Potential Therapeutic Target for Mesenchymal Stem Cell Expansion

Although pioneering preclinical research on the use of cell therapy for cardiac regeneration was conducted in the last quarter of the 20th century,1,2 a preponderance of advances have occurred in the 21st century, making this a relatively young field. In the first important clinical trial of cardiac cell therapy, begun in 2001, Menasche et al3 implanted autologous skeletal myoblasts into postinfarct scar at the time of coronary artery bypass surgery. Although the transplanted cells remained viable and exhibited contraction, they formed the nidus for serious ventricular tachyarrhythmias, which led to premature discontinuation of the trial. Despite this outcome, the trial energized the field, accelerating both preclinical and clinical research, albeit not with skeletal myoblasts. The extensive progress in cardiac regeneration is reviewed in this Compendium, and as occurs frequently in science, important observations have led to more questions and challenges (Table). View this table: Table. Important Challenges to Cell Therapy for Cardiac Regeneration Many cell types have been evaluated as candidates for cardiac regeneration. Among the earliest clinical trials, Zeiher’s group infused autologous bone marrow-derived progenitor cells into the coronary arteries of patients with acute,4 as well as healed myocardial infarction (MI)5 and reported improvements in left ventricular (LV) function. However, these results have not been fully confirmed by later studies, as pointed out in the review in the Compendium by Banarjee, Bolli, and Hare.6 Pittenger et al7 were among the first to direct attention to bone marrow-derived (stromal) mesenchymal stem cells (MSCs), emphasizing that these cells proliferated extensively in culture and suggesting that they could be attractive candidates for transplantation. In 2004, Chen et al8 reported that intracoronary infusion of autologous bone marrow-derived MSCs improved cardiac function. Zimmet and Hare pointed out that MSCs lack histocompatibility type II markers and elude rejection by …

In transplantation, hypothermic machine perfusion (HMP) has been shown to be superior to static cold storage (SCS) in terms of functional outcomes. Ex vivo machine perfusion offers the possibility to deliver drugs or other active substances, such as Mesenchymal Stem Cells (MSCs), directly into an organ without affecting the recipient. MSCs are multipotent, self-renewing cells with tissue-repair capacities, and their application to ameliorate ischemia- reperfusion injury (IRI) is being investigated in several preclinical and clinical studies. The aim of this study was to introduce MSCs into a translational model of hypothermic machine perfusion and to test the efficiency and feasibility of this method. Methods: three rodent kidneys, six porcine kidneys and three human kidneys underwent HMP with 1-5 × 106 labelled MSCs within respective perfusates. Only porcine kidneys were compared to a control group of 6 kidneys undergoing HMP without MSCs, followed by mimicked reperfusion with whole blood at 37 °C for 2 h for all 12 kidneys. Reperfusion perfusate samples were analyzed for levels of NGAL and IL-β by ELISA. Functional parameters, including urinary output, oxygen consumption and creatinine clearance, were compared and found to be similar between the MSC treatment group and the control group in the porcine model. IL-1β levels were higher in perfusate and urine samples in the MSC group, with a median of 285.3 ng/mL (IQR 224.3-407.8 ng/mL) vs. 209.2 ng/mL (IQR 174.9-220.1), p = 0.51 and 105.3 ng/mL (IQR 71.03-164.7 ng/mL) vs. 307.7 ng/mL (IQR 190.9-349.6 ng/mL), p = 0.16, respectively. MSCs could be traced within the kidneys in all models using widefield microscopy after HMP. The application of Mesenchymal Stem Cells in an ex vivo hypothermic machine perfusion setting is feasible, and MSCs can be delivered into the kidney grafts during HMP. Functional parameters during mimicked reperfusion were not altered in treated kidney grafts. Changes in levels of IL-1β suggest that MSCs might have an effect on the kidney grafts, and whether this leads to a positive or a negative outcome on IRI in transplantation needs to be determined in further experiments.

In a mouse model of menstruation stromal cells respond to breakdown of the tissue by changing their identity to become epithelial cells that are incorporated into the luminal epithelium which is rapidly 'healed' without scarring.

This study investigated whether there are marked differences in surface markers between rabbit and human mesenchymal stem cells (MSCs). Murine and rabbit MSCs have been reported to be CD90-negative. Rat MSCs have been reported to be CD71-negative. Our previous study also shows that rabbit MSCs are CD29-negative. However, human MSCs are generally considered to be CD29-, CD71-, and CD90-positive. Therefore, the surface markers of human MSCs might differ from those of other species. Rabbit bone marrow MSCs were obtained that had a multi-differentiation potential. The phenotype of these cells was studied using flow cytometry antibodies for 25 rabbit surface markers, namely, CD13, CD14, CD29, CD31, CD34, CD44, CD45, CD49d, CD49f, CD51, CD54, CD59, CD71, CD73, CD90, CD105, CD106, CD133, CD166, MHC I, MHC II, α-smooth muscle actin (α-SMA), cytokeratin, desmin, and vimentin. The phenotype of commercially available human MSCs was similarly studied using antibodies for human surface markers. CD14, CD31, CD34, CD45, CD49d, CD49f, CD51, CD54, CD71, CD106, CD133, MHC II, and cytokeratin were absent from both rabbit and human MSCs, while CD44, α-SMA, and vimentin were present on both cell lines. CD13, CD29, CD59, CD73, CD90, CD105, CD166, and MHC I were present on human MSCs, but not on rabbit MSCs. However, desmin was present on rabbit MSCs, but not on human MSCs. In total, the surface expression of nine markers differed between human and rabbit MSCs, whereas the surface expression of 16 markers was the same in the two cell lines.

In the August 2007 issue of Radiology Dr Schafer and colleagues (1) reported the upregulation of transferrin receptor (CD71) on rat mesenchymal stem cells (MSCs) labeled with superparamagnetic iron oxide nanoparticles (SPION).The authors should be commended for studying changes in the iron metabolism or storage to ensure safety of SPION cell labeling.However, their results and conclusions cannot be generalized to all SPION and transfection agents (TA) used for cells labeling.The authors report the upregulation of CD71 on late passage (i.e., 7-16) rat MSC after labeling with ferucarbotran.Several reports had shown that after multiple passages, human and mouse MSCs are prone to genetic instability with consequent malignant transformation (2,3).Later passage (> 5) is not biologically relevant since human (h) MSCs become senescent after 20-40 population doublings in culture (4).Human MSCs are positive for the transferrin receptor (5,6), however the authors showed no CD71 expression in control rat MSCs.Ferumoxidesprotamine sulfate labeled hMSCs have an expected transient decrease in CD71 mRNA and protein levels when compared to unlabeled cells (6).The upregulation of CD71 in the ferucarbotran labeled rat MSC (figure 3) cannot be extrapolated to human MSCs since control rat MSC do not express the transferrin receptor (1).The authors avoided comparing their findings to magnetic labeling of MSCs with ferumoxides and transfection agents (TA) such as poly-l-lysine (PLL) or protamine sulfate (Pro) (7-12).The authors report that the ferucarbotran complexed to DOSPER or jetPEI coated the cell surface but were not internalized.Ferumoxides complexed to PLL (12) or Pro (8,9) are located in endosomes in hMSCs and in other cells.The authors suggest labeling MSCs with SPION may affect homing capability of the stem cells.We and others have shown that ferumoxides-TA labeled hMSC will home to liver (7) tumors (11) or heart (13) by MRI and or by histology.The author's concern about the alteration in cell homing is not warranted especially when the SPION-TA complexes are localized in endosomes.Although the authors describe their study limitations, they overstate the clinical relevance and potential translation of their results.The author's advances in knowledge statement that SPIO-TA or USPIO-TA complexes only coats the MSC is not applicable to ferumoxides-TAs complexes and human MSC.The concerns raised by the author's are only pertinent to their study and are incorrect when ferumoxides is complexed to protamine sulfate.

Ischemic heart disease, especially myocardial infarction, is an important cause of heart failure. However, therapeutic potential of mesenchymal stem cells (MSC) for myocardial or pulmonary ischemia reperfusion (IR) injury is not well studied. Interleukin-10 (IL-10) is an anti-inflammatory cytokine. In this study MSC and IL-10 engineered MSC were tested for their ability to prevent IR injury and promote tissue repair. Methods: Bone marrow cells from Lewis rat were cultured in plastic tissue culture flasks for 72 hours; cells in suspension were discarded and the adherent cells (MSC) were expanded. MSC were transduced with r vIL-10-retrovirus and selected on neomycin (1mg/ml for 7 days). Experimental design: following 120 minutes of left lung ischemia, Group A, rats received vIL10 transduced MSC (∼15 x 10 6 ; i.v.); Group B, rats received empty vector engineered MSC; Group C, received MSC; and Group D, received saline. Group E received no ischemia or MSC. Left lung ischemia was performed by clamping left pulmonary artery/vein/bronchus at end inspiration. At 24h, 72 h, &amp; 7 days following ischemia, blood was collected from left and right pulmonary veins, separately. Results : Mean blood oxygenation (PaO2/FIO2 ratio, mmHg) at day 7 was significantly (P&lt;0.05) reduced following IR injury in Group B (161± 77) and Group D (181± 64), compared to MSC-vIL10 (404± 32; Group A) &amp; MSC (294±103;Group C) treated animals. In Group E, PaO2/FIO2 was 440± 42mm Hg. Significant improvement in lung oxygenation with MSC and vIL-10 therapy was observed as early as 3 days. MSC produced vIL-10 (∼5ng/ml) ex vivo . HP of lungs demonstrated reduced PMN cells with MSC therapy. Inflammatory mediators IL-1a, MCP-1, MIP-1a, and IL1-b protein levels were markedly increased in the injured lung; their analysis in MSC treated groups are underway. I.V. injected Tg GFP+MSC trafficked to the lung &amp; other tissues. VEGF &amp; HGF mRNA in MSC was 66% &amp; 43% of β-actin, respectively. Ex vivo expanded MSC were CD34-, CD45+ (5%), CD29+ (92%), CD80-, CD86+ (10%), CD90+ (∼90%), MHC Class I low &amp; MHC Class II-. Conclusions: Autologous MSC therapy prevented IR injury and served as an excellent vehicle to deliver cytokine. MSC therapy to prevent IR injury and enhance tissue regeneration in cardiopulmonary diseases seems promising.

Objectives. Tumor tissue produces abundant stromal cell-derived factor-1α (SDF-1α/CXCL12) that promotes recruitment of circulating stem/progenitor cells. We have recently found that SDF-1α induces elevated expression of CD162 and CD44, both are E-selectin ligands, on luminal endothelial cells lining capillaries. We thus postulate that highly expressed E-selectin ligands on tumor endothelium can serve as docking molecules to mediate specific homing of E-selectin-expressing circulating stem cells to tumor tissues. Here, we tested the role of E-selectin in mediating and facilitating homing of mesenchymal stem cells (MSC), which are modified to overexpress E-selectin, to SDF-1α−rich melanoma tissue. Methods. MSC from C57 BL6 and E-selectin-/- mice were enriched by culturing bone marrow cells in MesenCultTM medium. MSC were genetically labeled with luciferase (Luc2) or GFP in vitro. MSC from C57 BL6 mice were engineered to stably overexpress E-selectin (E-selectin+). Human melanoma cells were engineered to overexpress SDF-1α. 2 x 10^6 SDF-1α+/melanoma cells were pre-engrafted into NSG mice via tail-vein or subcu to form lung metastatic foci or skin melanoma, respectively. After 7-10 days, 1 x 10^6 E-selectin+ or E-selectin-/- MSC were injected into tumor-bearing mice through tail-vein or subcu, respectively. Homing of injected MSC to melanoma was assessed at various time points by IVIS (for Luc2+/MSC) and flow cytometry (for GFP+/MSC), respectively. Results. There were significantly increased numbers of engineered E-selectin-overexpressing MSC homing to melanoma tissues via systemic and local approaches, compared to control MSC or E-selectin-/-/MSC (p&amp;lt;0.05). Conclusions. E-selectin is an effective adhesion molecule in mediating tumor selective homing of MSC. Our study provides a useful method for targeted delivery of cells to tumor and may have therapeutic potential for developing stem cell-based cancer treatment. Citation Format: Cuixia Yang, Danjuan Li, Bo Wang, Omaida C. Velazquez, Zhao-Jun Liu. E-selectin mediates targeted delivery of mesenchymal stem cells to tumor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 194. doi:10.1158/1538-7445.AM2014-194

Mesenchymal stem cells (MSCs) ameliorate injury and accelerate repair in many organs, including the kidney, although the reparative mechanisms and interaction with macrophages have not been elucidated. This study investigated the reparative potential of human bone marrow-derived MSCs and traced their homing patterns following administration to mice with ischemia-reperfusion (IR) injury using whole body bioluminescence imaging. The effect of MSCs on macrophage phenotype following direct and indirect coculture was assessed using qPCR. Human cytokine production was measured using multiplex arrays. After IR, MSCs homed to injured kidneys where they afforded protection indicated by decreased proximal tubule kidney injury molecule-1 expression, blood urea nitrogen, and serum creatinine levels. SDS-PAGE and immunofluorescence labeling revealed MSCs reduced collagen α1(I) and IV by day 7 post-IR. Gelatin zymography confirmed that MSC treatment significantly increased matrix metalloproteinase-9 activity in IR kidneys, which contributed to a reduction in total collagen. Following direct and indirect coculture, macrophages expressed genes indicative of an anti-inflammatory "M2" phenotype. MSC-derived human GM-CSF, EGF, CXCL1, IL-6, IL-8, MCP-1, PDGF-AA, and CCL5 were identified in culture supernatants. In conclusion, MSCs home to injured kidneys and promote repair, which may be mediated by their ability to promote M2 macrophage polarization.

Cyclooxygenase (COX)-2 is generally known as an inducible enzyme, and it produces arachidonic acid to prostaglandin E2 (PGE2), which modulates bone metabolism. Here, we investigated the expression and role of COX isomers in human mesenchymal stem cells. Human mesenchymal stem cells constitutively expressed COX-2 as well as COX-1, and secretion of PGE2 was completely inhibited by NS-398, a specific inhibitor of COX-2. Levels of secreted PGE2 were strikingly higher in human mesenchymal stem cells than in osteoblastic cells differentiated from the mesenchymal cells. This higher production of PGE2 in mesenchymal stem cells was due to higher expression of membrane-associated PGE synthase (mPGES) regulated by early growth response factor-1 (Egr-1). Treatment of human mesenchymal stem cells with NS-398 suppressed expression of bone morphogenetic protein-2 (BMP-2). The suppression of BMP-2 by NS-398 was abrogated by an EP4 receptor agonist as well as by PGE2. Moreover, BMP-2 expression was suppressed by an EP4 receptor antagonist. These data indicate that PGE2 produced by COX-2 increases BMP-2 expression via binding the EP4 receptor.

Bone marrow derived mesenchymal stem cells (MSCs) hold great promise for treating immune disorders, although the immunoregulatory mechanisms remain largely controversial. Here we show that the mechanisms of MSC-mediated immunosuppression vary in different species. Under the same culture conditions, immunosuppression by MSCs derived from human or monkey was mediated by indoleamine 2, 3 dioxygenase (IDO), while immunosuppression by mouse MSCs depended on nitric oxide (NO). When the expression levels of inducible nitric oxide synthase (iNOS) and IDO were examined in mouse and human MSCs stimulated with their respective inflammatory cytokines, we found that human MSCs expressed high levels of IDO, but very low levels of iNOS, while mouse MSCs expressed high levels of iNOS and very little IDO. Except the executor molecules, moreover, the human and mouse MSCs shared some similarities. Like in mouse MSCs, immunosuppression by human MSCs was not intrinsic, but inducible by inflammatory cytokines. Additionally, IDO-mediated immunosuppression by human MSCs is also chemokine-dependent. These findings help to explain the apparently conflicting reports in the literatures and provide critical information to allow better application of human MSCs in treating immune disorders.

The objective of this study was to determine the effect of pulmonary intravascular macrophage depletion on systemic inflammation and ex vivo neutrophil apoptosis using an experimental model of intestinal ischemia and reperfusion injury in horses. Neutrophils were isolated before and after surgery from horses that were randomized to three treatment groups, namely, sham celiotomy (CEL, n = 4), intestinal ischemia and reperfusion (IR, n = 6), and intestinal ischemia and reperfusion with gadolinium chloride treatment to deplete pulmonary intravascular macrophages (PIMs, IRGC, n = 6). Neutrophil apoptosis was assessed with Annexin V and propidium iodide staining quantified with flow cytometry and caspase-3, caspase-8, and caspase-9 activities in neutrophil lysates. All horses experienced a systemic inflammatory response following surgery. Following surgery, ex vivo neutrophil apoptosis was significantly delayed after 12 or 24 h in culture, except in IRGC horses (12 h: CEL: P = 0.03, IR: P = 0.05, IRGC: P = 0.2; 24 h: CEL: P = 0.001, IR: P = 0.004, IRGC: P = 0.3). Caspase-3, caspase-8, and caspase-9 activities were significantly reduced in neutrophils isolated after surgery and cultured for 12 h in IR horses, but not in IRGC horses (IR caspase-3: P = 0.002, IR caspase-8: P = 0.002, IR caspase-9: P = 0.04). Serum TNF-α concentration was increased in IRGC horses for 6-18 h following jejunal ischemia. Following surgery, ex vivo equine neutrophil apoptosis was delayed via downregulation of caspase activity, which was ameliorated by PIM depletion potentially via upregulation of TNF-α.