Secretory products of DPSC mitigate inflammatory effects in microglial cells by targeting MAPK pathway.

Activated microglial cells in the central nervous system (CNS) are the main contributors to neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Inhibiting their activation will help in reducing inflammation and oxidative stress during pathogenesis, potentially limiting the progression of the diseases. The immunomodulation properties of dental pulp-derived stem cells (DPSC) make it a promising therapy for neurodegenerative disorders. This study aims to determine whether secretory factors of DPSC (DPSC℗) inhibit inflammation and proliferation of microglial cells and define the molecular mechanisms. Our quantitative RT-PCR analysis showed that the DPSC℗ reduced the markers of the inflammation and induced anti-inflammatory molecules in microglial cells. DPSC ℗ reduced the intracellular and mitochondrial reactive oxygen species (ROS) production and mitochondrial membrane potential in microglial cells. In addition, DPSC ℗ decreased the cellular bioenergetics parameters related to oxygen consumption rate (OCAR) and extracellular acidification rate (ECAR). We found that DPSC℗ inhibited microglial cell proliferation by activating a checkpoint molecule, Chk1 leading an arrest at the G1 phase of the cell cycle. To define the mechanism, we performed the western blot analysis and observed that the MAPK P38 pathway was inhibited by DPSC℗. Furthermore, a System biology analysis revealed that the BDNF and GDNF, secretory factors of DPSC, blocked at the phosphorylation site (Tyr 182) of the P38 molecule resulting in the inhibition of downstream signaling of inflammation. These data suggest that the DPSC℗ may be a potential therapeutic agent for neurodegenerative diseases.

KLF2 Regulates Neural Differentiation of Dental Pulp-derived Stem Cells by Modulating Autophagy and Mitophagy.

BACKGROUND: Transplantation of stem cells for treating neurodegenerative disorders is a promising future therapeutic approach. However, the molecular mechanism underlying the neuronal differentiation of dental pulp-derived stem cells (DPSC) remains inadequately explored. The current study aims to define the regulatory role of KLF2 (Kruppel-like factor 2) during the neural differentiation (ND) of DPSC. METHODS: We first investigated the transcriptional and translational expression of KLF2, autophagy, and mitophagy-associated markers during the ND of DPSC by using quantitative RT-PCR and western blot methods. After that, we applied the chemical-mediated loss- and gain-of-function approaches using KLF2 inhibitor, GGPP (geranylgeranyl pyrophosphate), and KLF2 activator, GGTI-298 (geranylgeranyl transferase inhibitor-298) to delineate the role of KLF2 during ND of DPSC. The western blot, qRT-PCR, and immunocytochemistry were performed to determine the molecular changes during ND after KLF2 deficiency and KLF2 sufficiency. We also analyzed the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) using the Seahorse XFe24 analyzer. RESULTS: Our study demonstrated that the expression level of KLF2, autophagy, and mitophagy-associated markers were significantly elevated during the ND of DPSC. Next, we found that the KLF2 inhibitor, GGPP significantly reduced the ND of DPSC. Inversely, KLF2 overexpression accelerated the molecular phenomenon of DPSC's commitment towards ND, indicating the crucial role of KLF2 in neurogenesis. Moreover, we found that the KLF2 positively regulated autophagy, mitophagy, and the Wnt5a signaling pathway during neurogenesis. Seahorse XFe24 analysis revealed that the ECAR and OCR parameters were significantly increased during ND, and inhibition of KLF2 marginally reversed them towards DPSC's cellular bioenergetics. However, KLF2 overexpression shifted the cellular energy metabolism toward the quiescent stage. CONCLUSION: Collectively, our findings provide the first evidence that the KLF2 critically regulates the neurogenesis of DPSC by inducing autophagy and mitophagy.

Secretome of Dental Pulp-Derived Stem Cells Reduces Inflammation and Proliferation of Glioblastoma Cells by Deactivating Mapk-Akt Pathway.

Background: Dental pulp-derived stem cells (DPSC) is a promising therapy as they modulate the immune response, so we evaluated the inhibitory effect of DPSC secretome (DPSC℗) on the proliferation and inflammation in human glioblastoma (GBM) cells (U-87 MG) and elucidated the concomitant mechanisms involved. Methods: The U87-MG cells were cultured with DPSC℗ for 24 h and assessed the expression of inflammatory molecules using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), generation of reactive oxygen species (ROS), and mitochondrial functionality using a seahorse flux analyzer. MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay and cell cycle analysis were performed to evaluate the proliferation and cell cycle. Finally, the protein levels were determined by western blot. Results: DPSC℗ reduced the inflammation and proliferation of U-87 MG cells by down-regulating the pro-inflammatory markers and up-regulating anti-inflammatory markers expressions through ROS-mediated signaling. Moreover, DPSC℗ significantly reduced the mitochondrial membrane potential (MMP) in the cells. The cellular bioenergetics revealed that all the parameters of oxygen consumption rate (OCAR) and the extracellular acidification rate (ECAR) were significantly decreased in the GBM cells after the addition of DPSC℗. Additionally, DPSC℗ decreased the GBM cell proliferation by arresting the cell cycle at the G1 phase through activation (phosphorylation) of checkpoint molecule CHK1. Furthermore, mechanistically, we found that the DPSC℗ impedes the phosphorylation of the mitogen-activated protein kinases (P38 MAPK) and protein kinase B (AKT) pathway. Conclusion: Our findings lend the first evidence of the inhibitory effects of DPSC℗ on proliferation and inflammation in GBM cells by altering the P38 MAPK-AKT pathway.

Epigallocatechin-3-gallate inhibits osteoclastic differentiation by modulating mitophagy and mitochondrial functions.

A natural plant product, epigallocatechin-3-gallate (EGCG), was evaluated for its effectiveness in the regulation of osteoclastogenesis. We found that EGCG inhibited the osteoclast (OC) differentiation in vitro, and in primary bone marrow cells in a dose-dependent manner. Quantitative RT-PCR studies showed that the EGCG reduced the expression of OC differentiation markers. DCFDA, MitoSOX, and JC-1 staining revealed that the EGCG attenuated the reactive oxygen species (ROS), and mitochondrial membrane potential; and flux analysis corroborated the effect of EGCG. We further found that the EGCG inhibited mRNA and protein expressions of mitophagy-related molecules. We confirmed that the OC differentiation was inhibited by EGCG by modulating mitophagy through AKT and p38MAPK pathways. Furthermore, in silico analysis revealed that the binding of RANK and RANKL was blocked by EGCG. Overall, we defined the mechanisms of osteoclastogenesis during arthritis for developing a new therapy using a natural compound besides the existing therapeutics.

Targeted Delivery of BZLF1 to DEC205 Drives EBV-Protective Immunity in a Spontaneous Model of EBV-Driven Lymphoproliferative Disease.

Epstein-Barr virus (EBV) is a human herpes virus that infects over 90% of the world's population and is linked to development of cancer. In immune-competent individuals, EBV infection is mitigated by a highly efficient virus-specific memory T-cell response. Risk of EBV-driven cancers increases with immune suppression (IS). EBV-seronegative recipients of solid organ transplants are at high risk of developing post-transplant lymphoproliferative disease (PTLD) due to iatrogenic IS. While reducing the level of IS may improve EBV-specific immunity and regression of PTLD, patients are at high risk for allograft rejection and need for immune-chemotherapy. Strategies to prevent PTLD in this vulnerable patient population represents an unmet need. We have previously shown that BZLF1-specific cytotoxic T-cell (CTL) expansion following reduced IS correlated with immune-mediated PTLD regression and improved patient survival. We have developed a vaccine to bolster EBV-specific immunity to the BZLF1 protein and show that co-culture of dendritic cells (DCs) loaded with a αDEC205-BZLF1 fusion protein with peripheral blood mononuclear cells (PMBCs) leads to expansion and increased cytotoxic activity of central-effector memory CTLs against EBV-transformed B-cells. Human-murine chimeric Hu-PBL-SCID mice were vaccinated with DCs loaded with αDEC205-BZLF1 or control to assess prevention of fatal human EBV lymphoproliferative disease. Despite a profoundly immunosuppressive environment, vaccination with αDEC205-BZLF1 stimulated clonal expansion of antigen-specific T-cells that produced abundant IFNγ and significantly prolonged survival. These results support preclinical and clinical development of vaccine approaches using BZLF1 as an immunogen to harness adaptive cellular responses and prevent PTLD in vulnerable patient populations.

The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer.

BACKGROUND: Breast cancer (BC) is the most common cancer in women and the leading cause of cancer-associated mortality in women. In particular, triple-negative BC (TNBC) has the highest rate of mortality due in large part to the lack of targeted treatment options for this subtype. Thus, there is an urgent need to identify new molecular targets for TNBC treatment. RALA and RALB are small GTPases implicated in growth and metastasis of a variety of cancers, although little is known of their roles in BC. METHODS: The necessity of RALA and RALB for TNBC tumor growth and metastasis were evaluated in vivo using orthotopic and tail-vein models. In vitro, 2D and 3D cell culture methods were used to evaluate the contributions of RALA and RALB during TNBC cell migration, invasion, and viability. The association between TNBC patient outcome and RALA and RALB expression was examined using publicly available gene expression data and patient tissue microarrays. Finally, small molecule inhibition of RALA and RALB was evaluated as a potential treatment strategy for TNBC in cell line and patient-derived xenograft (PDX) models. RESULTS: Knockout or depletion of RALA inhibited orthotopic primary tumor growth, spontaneous metastasis, and experimental metastasis of TNBC cells in vivo. Conversely, knockout of RALB increased TNBC growth and metastasis. In vitro, RALA and RALB had antagonistic effects on TNBC migration, invasion, and viability with RALA generally supporting and RALB opposing these processes. In BC patient populations, elevated RALA but not RALB expression is significantly associated with poor outcome across all BC subtypes and specifically within TNBC patient cohorts. Immunohistochemical staining for RALA in patient cohorts confirmed the prognostic significance of RALA within the general BC population and the TNBC population specifically. BQU57, a small molecule inhibitor of RALA and RALB, decreased TNBC cell line viability, sensitized cells to paclitaxel in vitro and decreased tumor growth and metastasis in TNBC cell line and PDX models in vivo. CONCLUSIONS: Together, these data demonstrate important but paradoxical roles for RALA and RALB in the pathogenesis of TNBC and advocate further investigation of RALA as a target for the precise treatment of metastatic TNBC.

Stromal Platelet-Derived Growth Factor Receptor-ß Signaling Promotes Breast Cancer Metastasis in the Brain.

Platelet-derived growth factor receptor-beta (PDGFRß) is a receptor tyrosine kinase found in cells of mesenchymal origin such as fibroblasts and pericytes. Activation of this receptor is dependent on paracrine ligand induction, and its preferred ligand PDGFB is released by neighboring epithelial and endothelial cells. While expression of both PDGFRß and PDGFB has been noted in patient breast tumors for decades, how PDGFB-to-PDGFRß tumor-stroma signaling mediates breast cancer initiation, progression, and metastasis remains unclear. Here we demonstrate this paracrine signaling pathway that mediates both primary tumor growth and metastasis, specifically, metastasis to the brain. Elevated levels of PDGFB accelerated orthotopic tumor growth and intracranial growth of mammary tumor cells, while mesenchymal-specific expression of an activating mutant PDGFRß (PDGFRßD849V) exerted proproliferative signals on adjacent mammary tumor cells. Stromal expression of PDGFRßD849V also promoted brain metastases of mammary tumor cells expressing high PDGFB when injected intravenously. In the brain, expression of PDGFRßD849V was observed within a subset of astrocytes, and aged mice expressing PDGFRßD849V exhibited reactive gliosis. Importantly, the PDGFR-specific inhibitor crenolanib significantly reduced intracranial growth of mammary tumor cells. In a tissue microarray comprised of 363 primary human breast tumors, high PDGFB protein expression was prognostic for brain metastases, but not metastases to other sites. Our results advocate the use of mice expressing PDGFRßD849V in their stromal cells as a preclinical model of breast cancer-associated brain metastases and support continued investigation into the clinical prognostic and therapeutic use of PDGFB-to-PDGFRß signaling in women with breast cancer. SIGNIFICANCE: These studies reveal a previously unknown role for PDGFB-to-PDGFRß paracrine signaling in the promotion of breast cancer brain metastases and support the prognostic and therapeutic clinical utility of this pathway for patients.See related article by Wyss and colleagues, p. 594.

Ferutinin directs dental pulp-derived stem cells towards the osteogenic lineage by epigenetically regulating canonical Wnt signaling.

Osteoporosis is a silent systemic disease that causes bone deterioration, and affects over 10 million people in the US alone. This study was undertaken to develop a potential stem cell therapy for osteoporosis. We have isolated and expanded human dental pulp-derived stem cells (DPSCs), characterized them, and confirmed their multipotential differentiation abilities. Stem cells often remain quiescent and require activation to differentiate and function. Herein, we show that ferutinin activates DPSCs by modulating the Wnt/ß-catenin signaling pathway and key osteoblast-secreted proteins osteocalcin and collagen 1A1 both mRNA and protein levels. To confirm that ferutinin modulates the Wnt pathway, we inhibited glycogen synthase kinase 3 (GSK3) and found that protein expression patterns were similar to those found in ferutinin-treated DPSCs. To evaluate the role of ferutinin in epigenetic regulation of canonical Wnt signaling, the pathway molecules Wnt3a and Dvl3 were analyzed using chromatin immunoprecipitation (ChIP)-quantitative PCR approaches. We confirmed that active marks of both H3K9 acetylation and H3K4 trimethylation were significantly enhanced in the promoter sites of the WNT3A and DVL3 genes in DPSCs after addition of ferutinin. These data provide evidence that ferutinin activates and promotes osteogenic differentiation of DPSCs, and could be used as an inducer as a potentially effective stem cell therapy for osteoporosis.

Myeloid Krüppel-Like Factor 2 Critically Regulates K/BxN Serum-Induced Arthritis.

Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease, and Krüppel-like factor 2 (KLF2) regulates immune cell activation and function. Herein, we show that in our experiments 50% global deficiency of KLF2 significantly elevated arthritic inflammation and pathogenesis, osteoclastic differentiation, matrix metalloproteinases (MMPs), and inflammatory cytokines in K/BxN serum-induced mice. The severities of RA pathogenesis, as well as the causative and resultant cellular and molecular factors, were further confirmed in monocyte-specific KLF2 deficient mice. In addition, induction of RA resulted in a decreased level of KLF2 in monocytes isolated from both mice and humans along with higher migration of activated monocytes to the RA sites in humans. Mechanistically, overexpression of KLF2 decreased the level of MMP9; conversely, knockdown of KLF2 increased MMP9 in monocytes along with enrichment of active histone marks and histone acetyltransferases on the MMP9 promoter region. These findings define the critical regulatory role of myeloid KLF2 in RA pathogenesis.

Nanofiber-expanded human CD34+ cells heal cutaneous wounds in streptozotocin-induced diabetic mice.

Despite advances in diabetic wound care, the significant number of amputations that occur every year demands more effective therapeutics. Herein, we offer an aminated polyethersulfone nanofiber-expanded human umbilical cord blood-derived CD34+ cells (henceforth CD34+ cells) effective therapy, tested in cutaneous wounds developed in streptozotocin-induced diabetic NOD/SCID mice. We show that systemic administration of CD34+ cells homed to the wound site and significantly accelerated wound closure. Wound closure was associated with improved re-epithelialization and increased neovascularization; and with decreased sustained pro-inflammatory activity of NF-κB and its downstream effector molecules TNF-α, IL-1ß, and IL-6 at the wound bed. This finding was further supported by the observation of a decreased number of myeloperoxidase positive neutrophils, and concomitantly increased levels of IL-10. In addition, improved granulation tissue formation was observed along with higher collagen deposition and myofibroblasts and decreased expressions of MMP-1. Mechanistically, CD34+ cells reduced the level of MMP-1 expression by inhibiting recruitment of NF-κB to the MMP-1 promoter site in dermal fibroblasts. In summary, we provide evidence of a novel nanofiber-expanded CD34+ stem cell therapeutic development for treating diabetic wounds by defining their cellular and molecular mechanisms.

Induction of Krüppel-like factor 2 reduces K/BxN serum-induced arthritis.

Krüppel-like factor 2 (KLF2) critically regulates activation and function of monocyte, which plays important pathogenic role in progressive joint destruction in rheumatoid arthritis (RA). It is yet to be established the molecular basis of KLF2-mediated regulation of monocytes in RA pathogenesis. Herein, we show that a class of compound, HDAC inhibitors (HDACi) induced KLF2 expression in monocytes both in vitro and in vivo. KLF2 level was also elevated in tissues, such as bone marrow, spleen and thymus in mice after infusion of HDACi. Importantly, HDACi significantly reduced osteoclastic differentiation of monocytes with the up-regulation of KLF2 and concomitant down-regulation of matrixmetalloproteinases both in the expression level as well as in the protein level. In addition, HDACi reduced K/BxN serum-induced arthritic inflammation and joint destruction in mice in a dose-dependent manner. Finally, co-immunoprecipitation and overexpression studies confirmed that KLF2 directly interacts with HDAC4 molecule in cells. These findings provide mechanistic evidence of KLF2-mediated regulation of K/BxN serum-induced arthritic inflammation.

Stromal PTEN determines mammary epithelial response to radiotherapy.

The importance of the tumor-associated stroma in cancer progression is clear. However, it remains uncertain whether early events in the stroma are capable of initiating breast tumorigenesis. Here, we show that in the mammary glands of non-tumor bearing mice, stromal-specific phosphatase and tensin homolog (Pten) deletion invokes radiation-induced genomic instability in neighboring epithelium. In these animals, a single dose of whole-body radiation causes focal mammary lobuloalveolar hyperplasia through paracrine epidermal growth factor receptor (EGFR) activation, and EGFR inhibition abrogates these cellular changes. By analyzing human tissue, we discover that stromal PTEN is lost in a subset of normal breast samples obtained from reduction mammoplasty, and is predictive of recurrence in breast cancer patients. Combined, these data indicate that diagnostic or therapeutic chest radiation may predispose patients with decreased stromal PTEN expression to secondary breast cancer, and that prophylactic EGFR inhibition may reduce this risk.

Retention of stemness and vasculogenic potential of human umbilical cord blood stem cells after repeated expansions on PES-nanofiber matrices.

Despite recent advances in cardiovascular medicine, ischemic diseases remain a major cause of morbidity and mortality. Although stem cell-based therapies for the treatment of ischemic diseases show great promise, limited availability of biologically functional stem cells mired the application of stem cell-based therapies. Previously, we reported a PES-nanofiber based ex vivo stem cell expansion technology, which supports expansion of human umbilical cord blood (UCB)-derived CD133(+)/CD34(+) progenitor cells ∼225 fold. Herein, we show that using similar technology and subsequent re-expansion methods, we can achieve ∼5 million-fold yields within 24 days of the initial seeding. Interestingly, stem cell phenotype was preserved during the course of the multiple expansions. The high level of the stem cell homing receptor, CXCR4 was expressed in the primary expansion cells, and was maintained throughout the course of re-expansions. In addition, re-expanded cells preserved their multi-potential differential capabilities in vitro, such as, endothelial and smooth muscle lineages. Moreover, biological functionality of the re-expanded cells was preserved and was confirmed by a murine hind limb ischemia model for revascularization. These cells could also be genetically modified for enhanced vasculogenesis. Immunohistochemical evidences support enhanced expression of angiogenic factors responsible for this enhanced neovascularization. These data further confirms that nanofiber-based ex-vivo expansion technology can generate sufficient numbers of biologically functional stem cells for potential clinical applications.

Induction of ATM/ATR pathway combined with Vγ2Vδ2 T cells enhances cytotoxicity of ovarian cancer cells.

Many ovarian cancer cells express stress-related molecule MICA/B on their surface that is recognized by Vγ2Vδ2 T cells through their NKG2D receptor, which is transmitted to downstream stress-signaling pathway. However, it is yet to be established how Vγ2Vδ2 T cell-mediated recognition of MICA/B signal is transmitted to downstream stress-related molecules. Identifying targeted molecules would be critical to develop a better therapy for ovarian cancer cells. It is well established that ATM/ATR signal transduction pathways, which is modulated by DNA damage, replication stress, and oxidative stress play central role in stress signaling pathway regulating cell cycle checkpoint and apoptosis. We investigated whether ATM/ATR and its down stream molecules affect Vγ2Vδ2 T cell-mediated cytotoxicity. Herein, we show that ATM/ATR pathway is modulated in ovarian cancer cells in the presence of Vγ2Vδ2 T cells. Furthermore, downregulation of ATM pathway resulted downregulation of MICA, and reduced Vγ2Vδ2 T cell-mediated cytotoxicity. Alternately, stimulating ATM pathway enhanced expression of MICA, and sensitized ovarian cancer cells for cytotoxic lysis by Vγ2Vδ2 T cells. We further show that combining currently approved chemotherapeutic drugs, which induced ATM signal transduction, along with Vγ2Vδ2 T cells enhanced cytotoxicity of resistant ovarian cancer cells. These findings indicate that ATM/ATR pathway plays an important role in tumor recognition, and drugs promoting ATM signaling pathway might be considered as a combination therapy together with Vγ2Vδ2 T cells for effectively treating resistant ovarian cancer cells.

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.

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-1ß, 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.

Hematopoietic stem cells improve dopaminergic neuron in the MPTP-mice.

Because of their ability for self-renewal and neural differentiation, stem cells are believed to be ideal for cell replacement therapy in Parkinson's disease (PD). Nanofiber-expanded human umbilical cord hematopoietic stem cells (HUHSCs) are advantageous to other stem cells as they provide a source of unlimited stem cell production for clinical application. In this study, we investigated whether 1. nanofiber-expanded HUHSCs are capable of neural differentiation in vitro, and 2. they could improve dopaminergic neuron morphology in the caudate/putamen (CPu) and substantia nigra pars compacta (SNc) of the MPTP-mouse model of PD. When cultured under neural differentiation conditions, nanofiber-expanded HUHSCs were able to undergo neural differentiation in vitro, as determined by gene and protein expression of neural markers such as MAP2, NeuN, HuC, GFAP and Oligo2. Thirty days after a single intracardioventricular injection of HUHSCs to MPTP-mice there was a significant recovery of tyrosine hydroxylase (TH) immunostaining in CPu. There was an increase in the size and staining density of TH+ cells in SNc, while their number was unchanged.

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.