Matthew Joseph

Stem Cell Therapy with Overexpressed VEGF and PDGF Genes Improves Cardiac Function in a Rat Infarct Model

Background Therapeutic potential was evaluated in a rat model of myocardial infarction using nanofiber-expanded human cord blood derived hematopoietic stem cells (CD133+/CD34+) genetically modified with VEGF plus PDGF genes (VIP). Methods and Findings Myocardial function was monitored every two weeks up to six weeks after therapy. Echocardiography revealed time dependent improvement of left ventricular function evaluated by M-mode, fractional shortening, anterior wall tissue velocity, wall motion score index, strain and strain rate in animals treated with VEGF plus PDGF overexpressed stem cells (VIP) compared to nanofiber expanded cells (Exp), freshly isolated cells (FCB) or media control (Media). Improvement observed was as follows: VIP>Exp> FCB>media. Similar trend was noticed in the exercise capacity of rats on a treadmill. These findings correlated with significantly increased neovascularization in ischemic tissue and markedly reduced infarct area in animals in the VIP group. Stem cells in addition to their usual homing sites such as lung, spleen, bone marrow and liver, also migrated to sites of myocardial ischemia. The improvement of cardiac function correlated with expression of heart tissue connexin 43, a gap junctional protein, and heart tissue angiogenesis related protein molecules like VEGF, pNOS3, NOS2 and GSK3. There was no evidence of upregulation in the molecules of oncogenic potential in genetically modified or other stem cell therapy groups. Conclusion Regenerative therapy using nanofiber-expanded hematopoietic stem cells with overexpression of VEGF and PDGF has a favorable impact on the improvement of rat myocardial function accompanied by upregulation of tissue connexin 43 and pro-angiogenic molecules after infarction.

Ex Vivo Nanofiber Expansion and Genetic Modification of Human Cord Blood-Derived Progenitor/Stem Cells Enhances Vasculogenesis

The stem cell therapy for treating ischemic diseases is promising; however, the limited availability and compromised quality of progenitor cells in aged and diseased patients limit its therapeutic use. Here we report a nanofiber-based ex vivo stem cell expansion technology and proangiogenic growth factors overexpression of human umbilical cord blood (UCB)-derived progenitor cells to enhance angiogenic potential of therapeutic stem cells. The progenitor cells were expanded ~225-fold on nanofiber-based serum-free ex vivo expansion culture technique without inducing differentiation. The expanded cells express high levels of stem cell homing receptor, CXCR4, and adhesion molecule, LFA-1. The nanofiber-expanded stem cells uptake AcLDL effectively, and migrate efficiently in an in vitro transmigration assay. These expanded cells can also differentiate into endothelial and smooth muscle cells in vitro. In a NOD/SCID mouse hind limb vascular injury model, nanofiber-expanded cells were more effective in blood flow restoration and this effect was further augmented by VEGF164 and PDGF-BB, growth factor overexpression. The data indicate that nanofiber-based ex vivo expansion technology can provide an essential number of therapeutic stem cells. Additionally, proangiogenic growth factors overexpression in progenitor cells can potentially improve autologous or allogeneic stem cell therapy for ischemic diseases.

Human Umbilical Cord Blood-Derived CD34+ Cells Reverse Osteoporosis in NOD/SCID Mice by Altering Osteoblastic and Osteoclastic Activities

Background Osteoporosis is a bone disorder associated with loss of bone mineral density and micro architecture. A balance of osteoblasts and osteoclasts activities maintains bone homeostasis. Increased bone loss due to increased osteoclast and decreased osteoblast activities is considered as an underlying cause of osteoporosis. Methods and Findings The cures for osteoporosis are limited, consequently the potential of CD34+ cell therapies is currently being considered. We developed a nanofiber-based expansion technology to obtain adequate numbers of CD34+ cells isolated from human umbilical cord blood, for therapeutic applications. Herein, we show that CD34+ cells could be differentiated into osteoblastic lineage, in vitro. Systemically delivered CD34+ cells home to the bone marrow and significantly improve bone deposition, bone mineral density and bone micro-architecture in osteoporotic mice. The elevated levels of osteocalcin, IL-10, GM-CSF, and decreased levels of MCP-1 in serum parallel the improvements in bone micro-architecture. Furthermore, CD34+ cells improved osteoblast activity and concurrently impaired osteoclast differentiation, maturation and functionality. Conclusions These findings demonstrate a novel approach utilizing nanofiber-expanded CD34+ cells as a therapeutic application for the treatment of osteoporosis.

Impact of Diffusion Barriers to Small Cytotoxic Molecules on the Efficacy of Immunotherapy in Breast Cancer

Molecular-focused cancer therapies, e.g., molecularly targeted therapy and immunotherapy, so far demonstrate only limited efficacy in cancer patients. We hypothesize that underestimating the role of biophysical factors that impact the delivery of drugs or cytotoxic cells to the target sites (for associated preferential cytotoxicity or cell signaling modulation) may be responsible for the poor clinical outcome. Therefore, instead of focusing exclusively on the investigation of molecular mechanisms in cancer cells, convection-diffusion of cytotoxic molecules and migration of cancer-killing cells within tumor tissue should be taken into account to improve therapeutic effectiveness. To test this hypothesis, we have developed a mathematical model of the interstitial diffusion and uptake of small cytotoxic molecules secreted by T-cells, which is capable of predicting breast cancer growth inhibition as measured both in vitro and in vivo. Our analysis shows that diffusion barriers of cytotoxic molecules conspire with γδ T-cell scarcity in tissue to limit the inhibitory effects of γδ T-cells on cancer cells. This may increase the necessary ratios of γδ T-cells to cancer cells within tissue to unrealistic values for having an intended therapeutic effect, and decrease the effectiveness of the immunotherapeutic treatment.

Umbilical cord blood-selected CD133+ cells exhibit vasculogenic functionality in vitro and in vivo

BACKGROUND AIMS Current clinical trials utilize non-selected bone marrow (BM) mononuclear cells (MNC) to augment vasculo genesis within ischemic vascular beds. Recent reports have identified a diminished number and function of hemat-opoietic stem cells (HSC) from aged and diseased patients. Umbilical cord blood (UCB) provides a potential robust allo-geneic source of HSC for therapeutic vasculogenesis. METHODS MNC and magnetically isolated CD133(+) cells were assessed for viability (trypan blue) and surface phenotype (flow cytometry). To test in vivo functionality of the cells, NOD/SCID mice underwent ligation of the right femoral artery followed immediately by cell injection. Blood flow recovery, necrosis, BM engraftment of human cells and histologic capillary density were determined. Cells were tested for potential mechanisms mediating the in vivo effects, including migration, cytokine secretion and angiogenic augmentation (Matrigel assays). RESULTS Surface expression analysis showed CD31 (PECAM) expression was greatly increased in UCB CD133(+) cells compared with BM MNC. At 28 days, perfusion ratios were highest in animals receiving UCB CD133(+) cells, while animals receiving BM CD133(+) cells and BM MNC demonstrated perfusion ratios statistically higher than in animals treated with cytokine media alone. Animals receiving CD133(+) cells showed a statistically higher capillary density, reduced severe digit necrosis and increased engraftment in the BM than animals treated with unselected BM MNC. In vitro studies showed equivalent migration to stromal-derived factor-1 (SDF-1), increased production of tumor necrosis factor alpha (TNF-alpha) and increased branch points with the co-incubation of CD133(+) cells with human umbilical vein endothelial cells (HUVEC) in the Matrigel angiogenesis assay. CONCLUSIONS Taken together, UCB CD133(+) cells exhibit robust vasculogenic functionality compared with BM MNC in response to ischemia.

Human Ovarian Tumor Cells Escape γδ T Cell Recognition Partly by Down Regulating Surface Expression of MICA and Limiting Cell Cycle Related Molecules

Background Mechanisms of human Vγ2Vδ2 T cell-mediated tumor immunity have yet to be fully elucidated. Methods and Findings At least some tumor cell recognition is mediated by NKG2D-MICA interactions. Herein, by using MTT assay and PI-BrdU co-staining and Western-blot, we show that these Vγ2Vδ2 T cells can limit the proliferation of ovarian tumor cells by down regulation of apoptosis and cell cycle related molecules in tumor cells. Cell-to-cell contact is critical. γδ T cell-resistant, but not susceptible ovarian tumor cells escape γδ T cell-mediated immune recognition by up-regulating pErk1/2, thereby decreasing surface MICA levels. Erk1/2 inhibitor pretreatment or incubation prevents this MICA decrease, while up-regulating key cell cycle related molecules such as CDK2, CDK4 and Cyclin D1, as well as apoptosis related molecules making resistant tumor cells now vulnerable to γδ T cell-mediated lysis. Conclusion These findings demonstrate novel effects of γδT cells on ovarian tumor cells.

Human Vγ2Vδ2 T cells limit breast cancer growth by modulating cell survival-, apoptosis-related molecules and microenvironment in tumors.

Innate immune system has been known to play an important role in inhibiting the malignant transformation, tumor progression and invasion. However, the mechanistic basis remains ambiguous. Despite polyclonality of human γδ T cells, Vγ2Vδ2 T cell subset was shown to recognize and limit the growth of various tumors at various degrees. The differential recognition of the tumor cells by Vγ2Vδ2 T cells are yet to be defined. Our study reveals that γδ T cells limit in vitro growth of most breast tumor cells, such as SkBr7 (HER2+), MCF7 (ER+) and MDA‐MB‐231 (ER−) by inhibiting their survival and inducing apoptosis, except BrCa‐MZ01 (PR+) cells. To investigate detail mechanisms of antineoplastic effects, we found that cell death was associated with the surface expression levels of MICA/B and ICAM1. Molecular signaling analysis demonstrated that inhibition of cell growth by γδ T cells was associated with the lower expression levels of cell survival‐related molecules such as AKT, ERK and concomitant upregulation of apoptosis‐related molecules, such as PARP, cleaved caspase 3 and tumor suppressor genes PTEN and P53. However, opposite molecular signaling was observed in the resistant cell line after coculture with γδ T cells. In vivo, antineoplastic effects of γδ T cells were also documented, where tumor growth was inhibited due to the downregulation of survival signals, strong induction of apoptotic molecules, disruption of microvasculature and increased infiltration of tumor associated macrophages. These findings reveal that a complex molecular signaling is involved in γδ T cell‐mediated antineoplastic effects.

Nanofiber-expanded human umbilical cord blood–derived CD34+ cell therapy accelerates cutaneous wound closure in NOD/SCID mice

Nanofiber‐expanded human umbilical cord blood–derived CD34+ cell therapy has been shown to have potential applications for peripheral and myocardial ischaemic diseases. However, the efficacies of expanded CD34+ cell therapy for treating cutaneous wounds and its mechanisms of action have yet to be established. Using an excisional wound model in non‐obese diabetic/severe combined immune deficient mice, we show herein that CD34+ cells accelerate the wound‐healing process by enhancing collagen synthesis, and increasing fibroblast cell migration within the wound bed. Concomitantly, reduced levels of matrix metalloproteinase (MMPs) such as MMP1, MMP3, MMP9 and MMP13 were detected in the wound beds of animals treated with CD34+ cells compared with vehicle‐treated controls. CD34+ cells were found to mediate enhanced migration and proliferation of dermal fibroblast cells in vitro. Moreover, CD34+ cells secrete collagen in a serum‐deprived environment. In mechanistic studies, co‐culture of CD34+ cells with primary skin fibroblasts increased the expression of collagen1A1, a component of type 1 collagen, and decreased the expression of MMP1 in fibroblast cells in the presence of a proteasome inhibitor. Finally, CD34+ cell–mediated functions were transcriptionally regulated by the c‐Jun N‐terminal kinases pathway. Collectively, these data provide evidence of therapeutic efficacy and a novel mechanism of nanofiber‐expanded CD34+ cell–mediated accelerated wound healing.

Kruppel-like factor 2 (KLF2) regulates monocyte differentiation and functions in mBSA and IL-1β-induced arthritis.

Kruppel-like factor 2 (KLF2) plays an important role in the regulation of a variety of immune cells, including monocytes. We have previously shown that KLF2 inhibits proinflammatory activation of monocytes. However, the role of KLF2 in arthritis is yet to be investigated. In the current study, we show that recruitment of significantly greater numbers of inflammatory subset of CD11b(+)F4/80(+)Ly6C+ monocytes to the inflammatory sites in KLF2 hemizygous mice compared to the wild type littermate controls. In parallel, inflammatory mediators, MCP-1, Cox-2 and PAI-1 were significantly up-regulated in bone marrow-derived monocytes isolated from KLF2 hemizygous mice, in comparison to wild-type controls. Methylated-BSA and IL-1β-induced arthritis was more severe in KLF2 hemizygous mice as compared to the littermate wild type controls. Consistent with this observation, monocytes isolated from KLF2 hemizygous mice showed an increased number of cells matured and differentiated towards osteoclastic lineage, potentially contributing to the severity of cartilage and bone damage in induced arthritic mice. The severity of arthritis was associated with the higher expression of proteins such as HSP60, HSP90 and MMP13 and attenuated levels of pPTEN, p21, p38 and HSP25/27 molecules in bone marrow cells of arthritic KLF2 hemizygous mice compared to littermate wild type controls. The data provide new insights and evidences of KLF2-mediated transcriptional regulation of arthritis via modulation of monocyte differentiation and function.

Nanofiber-expanded human umbilical cord blood-derived CD34 + cell therapy accelerates murine cutaneous wound closure by attenuating pro-inflammatory factors and secreting IL-10

Nanofiber-expanded human umbilical cord blood-derived CD34+ cell therapy is under consideration for treating peripheral and cardiac ischemia. However, the therapeutic efficacy of nanofiber-expanded human umbilical cord blood-derived (NEHUCB) CD34+ cell therapy for wound healing and its mechanisms are yet to be established. Using an excision wound model in NOD/SCID mice, we show herein that NEHUCB-CD34+ cells home to the wound site and significantly accelerate the wound-healing process compared to vehicle-treated control. Histological analysis reveals that accelerated wound closure is associated with the re-epithelialization and increased angiogenesis. Additionally, NEHUCB-CD34+ cell-therapy decreases expression of pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6 and NOS2A in the wound bed, and concomitantly increases expression of IL-10 compared to vehicle-treated control. These findings were recapitulated in vitro using primary dermal fibroblasts and NEHUCB-CD34+ cells. Moreover, NEHUCB-CD34+ cells attenuate NF-κB activation and nuclear translocation in dermal fibroblasts through enhanced secretion of IL-10, which is known to bind to NF-κB and suppress transcriptional activity. Collectively, these data provide novel mechanistic evidence of NEHUCB-CD34+ cell-mediated accelerated wound healing.