Won Il Oh

Mesenchymal stem cells for bronchopulmonary dysplasia: phase 1 dose-escalation clinical trial.

OBJECTIVE To assess the safety and feasibility of allogeneic human umbilical cord blood (hUCB)-derived mesenchymal stem cell (MSC) transplantation in preterm infants. STUDY DESIGN In a phase I dose-escalation trial, we assessed the safety and feasibility of a single, intratracheal transplantation of hUCB-derived MSCs in preterm infants at high risk for bronchopulmonary dysplasia (BPD). The first 3 patients were given a low dose (1 × 10(7) cells/kg) of cells, and the next 6 patients were given a high dose (2 × 10(7) cells/kg). We compared their adverse outcomes, including BPD severity, with those of historical case-matched comparison group. RESULTS Intratracheal MSC transplantation was performed in 9 preterm infants, with a mean gestational age of 25.3 ± 0.9 weeks and a mean birth weight of 793 ± 127 g, at a mean of 10.4 ± 2.6 days after birth. The treatments were well tolerated, without serious adverse effects or dose-limiting toxicity attributable to the transplantation. Levels of interleukin-6, interleukin-8, matrix metalloproteinase-9, tumor necrosis factor α, and transforming growth factor β1 in tracheal aspirates at day 7 were significantly reduced compared with those at baseline or at day 3 posttransplantation. BPD severity was lower in the transplant recipients, and rates of other adverse outcomes did not differ between the comparison group and transplant recipients. CONCLUSION Intratracheal transplantation of allogeneic hUCB-derived MSCs in preterm infants is safe and feasible, and warrants a larger and controlled phase II study.

Timing of Umbilical Cord Blood Derived Mesenchymal Stem Cells Transplantation Determines Therapeutic Efficacy in the Neonatal Hyperoxic Lung Injury

Intratracheal transplantation of human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) attenuates the hyperoxia-induced neonatal lung injury. The aim of this study was to optimize the timing of MSCs transplantation. Newborn Sprague-Dawley rats were randomly exposed to hyperoxia (90% for 2 weeks and 60% for 1 week) or normoxia after birth for 21 days. Human UCB-derived MSCs (5×105 cells) were delivered intratracheally early at postnatal day (P) 3 (HT3), late at P10 (HT10) or combined early+late at P3+10 (HT3+10). Hyperoxia-induced increase in mortality, TUNEL positive cells, ED1 positive alveolar macrophages, myeloperoxidase activity and collagen levels, retarded growth and reduced alveolarization as evidenced by increased mean linear intercept and mean alveolar volume were significantly better attenuated in both HT3 and HT3+10 than in HT10. Hyperoxia-induced up-regulation of both cytosolic and membrane p47phox indicative of oxidative stress, and increased inflammatory markers such as tumor necrosis factor-α, interleukin (IL) -1α, IL-1β, IL-6, and transforming growth factor-β measured by ELISA, and tissue inhibitor of metalloproteinase-1, CXCL7, RANTES, L-selectin and soluble intercellular adhesion molecule-1 measured by protein array were consistently more attenuated in both HT3 and HT3+10 than in HT10. Hyperoxia-induced decrease in hepatocyte growth factor and vascular endothelial growth factor was significantly up-regulated in both HT3 and HT3+10, but not in HT10. In summary, intratracheal transplantation of human UCB derived MSCs time-dependently attenuated hyperoxia-induced lung injury in neonatal rats, showing significant protection only in the early but not in the late phase of inflammation. There were no synergies with combined early+late MSCs transplantation.

Mesenchymal Stem Cells Prevent Hydrocephalus After Severe Intraventricular Hemorrhage

Background and Purpose— Severe intraventricular hemorrhage (IVH) in premature infants and the ensuing posthemorrhagic hydrocephalus cause significant mortality and neurological disabilities, and there are currently no effective therapies. This study determined whether intraventricular transplantation of human umbilical cord blood-derived mesenchymal stem cells prevents posthemorrhagic hydrocephalus development and attenuates brain damage after severe IVH in newborn rats. Methods— To induce severe IVH, 100 &mgr;L of blood was injected into each lateral ventricle of postnatal day 4 (P4) Sprague-Dawley rats. Human umbilical cord blood-derived mesenchymal stem cells or fibroblasts (1×105) were transplanted intraventricularly under stereotaxic guidance at P6. Serial brain MRI and behavioral function tests, such as the negative geotaxis test and rotarod test, were performed. At P32, brain tissue and cerebrospinal fluid were obtained for histological and biochemical analyses. Results— Intraventricular transplantation of umbilical cord blood-derived mesenchymal stem cells, but not fibroblasts, prevented posthemorrhagic hydrocephalus development and significantly attenuated impairment on behavioral tests; the increased terminal deoxynycleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling-positive cells; increased expression of inflammatory cytokines, such as interleukin-1&agr;, interleukin-1&bgr;, interleukin-6, and tumor necrosis factor-&agr;; increased astrogliosis; and reduced corpus callosal thickness and myelin basic protein expression after inducing severe IVH. Conclusions— Intraventricular transplantation of umbilical cord blood-derived mesenchymal stem cells significantly attenuated the posthemorrhagic hydrocephalus and brain injury after IVH. This neuroprotective mechanism appears to be mediated by the anti-inflammatory effects of these cells.

Impact of Myocardial Infarct Proteins and Oscillating Pressure on the Differentiation of Mesenchymal Stem Cells : Effect of Acute Myocardial Infarction on Stem Cell Differentiation

Stem cell transplantation in acute myocardial infarction (AMI) has emerged as a promising therapeutic option. We evaluated the impact of AMI on mesenchymal stem cell (MSC) differentiation into cardiomyocyte lineage. Cord blood‐derived human MSCs were exposed to in vitro conditions simulating in vivo environments of the beating heart with acute ischemia, as follows: (a) myocardial proteins or serum obtained from sham‐operated rats, and (b) myocardial proteins or serum from AMI rats, with or without application of oscillating pressure. Expression of cardiac‐specific markers on MSCs was greatly induced by the infarcted myocardial proteins, compared with the normal proteins. It was also induced by application of oscillating pressure to MSCs. Treatment of MSCs with infarcted myocardial proteins and oscillating pressure greatly augmented expression of cardiac‐specific genes. Such expression was blocked by inhibitor of transforming growth factor β1 (TGF‐β1) or bone morphogenetic protein‐2 (BMP‐2). In vitro cellular and electrophysiologic experiments showed that these differentiated MSCs expressing cardiomyocyte‐specific markers were able to make a coupling with cardiomyocytes but not to selfbeat. The pathophysiologic significance of in vitro results was confirmed using the rat AMI model. The protein amount of TGF‐β1 and BMP‐2 in myocardium of AMI was significantly higher than that in normal myocardium. When MSCs were transplanted to the heart and analyzed 8 weeks later, they expressed cardiomyocyte‐specific markers, leading to improved cardiac function. These in vitro and in vivo results suggest that infarct‐related biological and physical factors in AMI induce commitment of MSCs to cardiomyocyte‐like cells through TGF‐β/BMP‐2 pathways.

Critical Role of Vascular Endothelial Growth Factor Secreted by Mesenchymal Stem Cells in Hyperoxic Lung Injury

Intratracheal transplantation of human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) protects against neonatal hyperoxic lung injury by a paracrine rather than a regenerative mechanism. However, the role of paracrine factors produced by the MSCs, such as vascular endothelial growth factor (VEGF), has not been delineated. This study examined whether VEGF secreted by MSCs plays a pivotal role in protecting against neonatal hyperoxic lung injury. VEGF was knocked down in human UCB-derived MSCs by transfection with small interfering RNA specific for human VEGF. The in vitro effects of MSCs with or without VEGF knockdown or neutralizing antibody were evaluated in a rat lung epithelial (L2) cell line challenged with H2O2. To confirm these results in vivo, newborn Sprague-Dawley rats were exposed to hyperoxia (90% O2) for 14 days. MSCs (1 × 10(5) cells) with or without VEGF knockdown were administered intratracheally at postnatal Day 5. Lungs were serially harvested for biochemical and histologic analyses. VEGF knockdown and antibody abolished the in vitro benefits of MSCs on H2O2-induced cell death and the up-regulation of inflammatory cytokines in L2 cells. VEGF knockdown also abolished the in vivo protective effects of MSCs in hyperoxic lung injury, such as the attenuation of impaired alveolarization and angiogenesis, reduction in the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive and ED-1-positive cells, and down-regulation of proinflammatory cytokine levels. Our data indicate that VEGF secreted by transplanted MSCs is one of the critical paracrine factors that play seminal roles in attenuating hyperoxic lung injuries in neonatal rats.

Cytotoxicity of human umbilical cord blood-derived mesenchymal stem cells against human malignant glioma cells

BackgroundMesenchymal stem cells (MSCs) represent a potential useful source for cell-based glioma therapies because these cells evidence both orthodox and unorthodox plasticity and also show tropism for cancer. In this study, the authors attempted to access the cytotoxicity of human umbilical cord blood (hUCB)-derived MSCs, with or without cytokine activations against malignant glioma cells.Materials and methodshUCB-derived MSCs were activated by interleukin-2, interleukin-15, granulocyte macrophage colony-stimulating factor, and combinations. The hUCB-derived MSCs and activated hUCB-derived MSCs were effector cells. The cytotoxicity of the unactivated hUCB-derived MSCs and activated hUCB-derived MSCs against the target cells (human malignant glioma cells) was estimated via visual survival cell assays and transwell inserts. Phenotypic changes occurring in these hUCB-derived MSCs before and after cytokine activation were determined via flow cytometry. The secreted proteins from these effector cells were estimated via enzyme-linked immunosorbent assays.ResultsWe noted a significant cytotoxicity of hUCB-derived MSCs against malignant glioma cells. In addition, the hUCB-derived MSCs activated with cytokines evidenced significantly higher cytotoxicity than that observed with unactivated hUCB-derived MSCs. Differentiated immune effectors cells from the hUCB-derived MSCs after cytokine activation were not shown to have increased in number. However, the activated hUCB-derived MSCs secreted more immune response-related proteins (interleukin 4, interferon-γ) than did the unactivated hUCB-derived MSCs.ConclusionThe data collected herein confirm for the first time that hUCB-derived MSCs, with or without activation, evidence significant cytotoxicity against human malignant glioma cells, and the immune response-related proteins secreted in this process may perform relevant functions.

Human Umbilical Cord Blood-Derived Mesenchymal Stem Cell Therapy Promotes Functional Recovery of Contused Rat Spinal Cord through Enhancement of Endogenous Cell Proliferation and Oligogenesis

Numerous studies have shown the benefits of mesenchymal stem cells (MSCs) on the repair of spinal cord injury (SCI) model and on behavioral improvement, but the underlying mechanisms remain unclear. In this study, to investigate possible mechanisms by which MSCs contribute to the alleviation of neurologic deficits, we examined the potential effect of human umbilical cord blood-derived MSCs (hUCB-MSCs) on the endogenous cell proliferation and oligogenesis after SCI. SCI was injured by contusion using a weight-drop impactor and hUCB-MSCs were transplanted into the boundary zone of the injured site. Animals received a daily injection of bromodeoxyuridine (BrdU) for 7 days after treatment to identity newly synthesized cells of ependymal and periependymal cells that immunohistochemically resembled stem/progenitor cells was evident. Behavior analysis revealed that locomotor functions of hUCB-MSCs group were restored significantly and the cavity volume was smaller in the MSCs-transplanted rats compared to the control group. In MSCs-transplanted group, TUNEL-positive cells were decreased and BrdU-positive cells were significantly increased rats compared with control group. In addition, more of BrdU-positive cells expressed neural stem/progenitor cell nestin and oligo-lineage cell such as NG2, CNPase, MBP and glial fibrillary acidic protein typical of astrocytes in the MSC-transplanted rats. Thus, endogenous cell proliferation and oligogenesis contribute to MSC-promoted functional recovery following SCI.

Long-Term (Postnatal Day 70) Outcome and Safety of Intratracheal Transplantation of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells in Neonatal Hyperoxic Lung Injury

Purpose This study was performed to evaluate the long-term effects and safety of intratracheal (IT) transplantation of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in neonatal hyperoxic lung injury at postnatal day (P)70 in a rat model. Materials and Methods Newborn Sprague Dawley rat pups were subjected to 14 days of hyperoxia (90% oxygen) within 10 hours after birth and allowed to recover at room air until sacrificed at P70. In the transplantation groups, hUCB-MSCs (5×105) were administered intratracheally at P5. At P70, various organs including the heart, lung, liver, and spleen were histologically examined, and the harvested lungs were assessed for morphometric analyses of alveolarization. ED-1, von Willebrand factor, and human-specific nuclear mitotic apparatus protein (NuMA) staining in the lungs and the hematologic profile of blood were evaluated. Results Impaired alveolar and vascular growth, which evidenced by an increased mean linear intercept and decreased amount of von Willebrand factor, respectively, and the hyperoxia-induced inflammatory responses, as evidenced by inflammatory foci and ED-1 positive alveolar macrophages, were attenuated in the P70 rat lungs by IT transplantation of hUCB-MSCs. Although rare, donor cells with human specific NuMA staining were persistently present in the P70 rat lungs. There were no gross or microscopic abnormal findings in the heart, liver, or spleen, related to the MSCs transplantation. Conclusion The protective and beneficial effects of IT transplantation of hUCB-MSCs in neonatal hyperoxic lung injuries were sustained for a prolonged recovery period without any long-term adverse effects up to P70.

N-cadherin Determines Individual Variations in the Therapeutic Efficacy of Human Umbilical Cord Blood-derived Mesenchymal Stem Cells in a Rat Model of Myocardial Infarction

In this study, we established and characterized human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) from four different donors. However, the hUCB-MSCs showed remarkable variations in their therapeutic efficacy for repairing rat infarcted myocardium (including the process of angiogenesis) 8 weeks after transplantation. In addition, we observed that the level of vascular endothelial growth factor (VEGF) is correlated with the therapeutic efficacy of the four hUCB-MSCs. Next, to investigate the practical application of hUCB-MSCs, we searched for surface signature molecules that could serve as indicators of therapeutic efficacy. The gene for N-cadherin was the only cell surface gene that was highly expressed in the most effective hUCB-MSCs, both at the transcriptional and translational levels. We observed downregulation and upregulation of VEGF in response to N-cadherin blocking and N-cadherin overexpression, respectively. Activation of extracellular signal-regulated kinase (ERK), but not protein kinase B, was increased when N-cadherin expression was increased, whereas disruption of N-cadherin-mediated cell-cell contact induced suppression of ERK activation and led to VEGF downregulation. Moreover, by investigating hUCB-MSCs overexpressing N-cadherin or N-cadherin knockdown hUCB-MSCs, we confirmed the in vivo function of N-cadherin. In addition, we observed that DiI-labeled hUCB-MSCs express N-cadherin in the peri-infarct area and interact with cardiomyocytes.

Cotransplanted bone marrow derived mesenchymal stem cells (MSC) enhanced engraftment of hematopoietic stem cells in a MSC-dose dependent manner in NOD/SCID mice.

Transplantation of marrow-derived mesenchymal stem cells (MSCs), expanded by culture in addition to whole bone marrow, has been shown to enhance engraftment of human hematopoietic stem cells (HSCs). Our hypothesis was that there might be an optimum ratio range that could enhance engraftment. We examined the percent donor chimerism according to the ratio of HSCs to MSCs in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. We tested a series of ratios of co-transplanted CD34+-selected bone marrow cells, and marrow-derived MSCs into sublethally irradiated NOD/SCID mice. In all experiments, 1×105 bone marrow derived human CD34+ cells were administered to each mouse and human MSCs from different donors were infused concomitantly. We repeated the procedure three times and evaluated engraftment with flow cytometry four weeks after each transplantation. Serial ratios of HSCs to MSCs were 1:0, 1:1, 1:2 and 1:4, in the first experiment, 1:0, 1:1, 1:2, 1:4 and 1:8 in the second and 1:0, 1:1, 1:4, 1:8 and 1:16 in the third. Cotransplantation of HSCs and MSCs enhanced engraftment as the dose of MSCs increased. Our results suggest that the optimal ratio of HSCs and MSCs for cotransplantation might be in the range of 1:8-1:16; whereas, an excessive dose of MSCs might decrease engraftment efficiency.