ROLE OF STAT3 IN KERATINOCYTE STEM CELLS DURING SKIN TUMORIGENESIS

STATs play crucial roles in a wide variety of biological functions, including development, proliferation, differentiation, migration and in cancer development. In the present study, we examined the impact of Stat3 deletion or activation on behavior of keratinocytes, including keratinocyte stem cells (KSCs). Deletion of Stat3 specifically in the bulge region of the hair follicle using K15.CrePR1 X Stat3fl/fl mice led to decreased tumor development by altering survival of bulge region KSCs. To further understand the role of KSCs in skin tumorigenesis, K5.Stat3C transgenic (Tg) mice which express a constitutively active/dimerized form of Stat3 called Stat3C via the bovine keratin 5 (K5) promoter were studied. The number of CD34 and α6 integrin positive cells was significantly reduced in Tg mice as compared to non-transgenic (NTg) littermates. There was a concomitant increase in the progenitor populations (Lgr-6, Lrig-1 and Sca-1) in the Tg mice vs. the stem cell population (CD34 and Keratin15). To investigate the mechanism underlying the increase in the progenitor population at the expense of bulge region KSCs we examined if Stat3C expression was involved in inducing migration of the bulge region KSCs. There was altered β-catenin and α6-integrin expression in the hair follicles of Tg mice, which may have contributed to reduced adhesive interactions between the epithelial cells and the basement membrane facilitating migration out of the niche. To further study the effect of Stat3 on differentiation of keratinocytes we analyzed the epidermal keratinocytes in K5.Cre X Stat3fl/fl mice. There was an increase in the expression of epidermal differentiation markers in the Stat3 knockout mice. These data suggest that deletion of Stat3 in the epidermis and hair follicle induced differentiation in these cells. Preliminary studies done with the BK5.Stat3C mouse model suggests that multiple hair follicle stem/progenitor populations may be involved in skin tumor development and progression in this model of skin tumorigenesis. Overall, these data suggest that Stat3 plays an important role in differentiation as well as migration of keratinocytes and that these effects may play a role during epithelial carcinogenesis.

ELUCIDATING THE ROLE OF CD44 EXPRESSION ON MESENCHYMAL STEM CELLS WITHIN THE TUMOR MICROENVIRONMENT

The tumor microenvironment is comprised of a vast array of heterogeneous cells including both normal and neoplastic cells. The tumor stroma recruitment process has been exploited for an effective gene delivery technique using bone marrow derived MSC. Targeted migration of the MSC toward the tumor microenvironment, while successful, is not yet fully understood. This study was designed to assess the role of CD44 in the migration of MSC toward the tumor microenvironment and to determine the implications of CD44-deficient MSC within the tumor stroma. Inhibition of MSC migration was evaluated through a variety of methods in vitro and in vivo including CD44 receptor knockdown, CD44 antagonists, CD44 neutralizing antibodies and small molecule inhibitor of matrix metalloproteinases. Blocking CD44 signaling through MMP inhibition was characterized by lack of intracellular domain cleavage and lead to the decrease in Twist gene expression. A functional relationship between CD44 and Twist expression was confirmed by chromatin immunoprecipitation. Next, a series of murine tumor models were used to examine the role of CD44 deficient stroma within the tumor microenvironment. Labeled transgenic CD44 knockout (KO) MSC or wild type (WT) C57/B6 MSC were used to analyze the stromal incorporation within murine breast carcinomas (EO771 and 4T1). Subsequent tumors were analyzed for vessel formation (CD31), and the presence of tumor associated fibroblast (TAF) markers, α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), and fibroblast specific protein (FSP). The tumors with CD44KO MSC cells had less vessel formation than the tumors with WT MSC. The lack of fibroblastic TAF population as defined by FAP/FSP expression by the CD44KO MSC admixed tumors suggest that the bone marrow derived population of MSC were unable to contribute to the fibroblastic stromal population. Subsequently, a bone marrow transplantation experiment confirmed the endogenous migratory deficiencies of the CD44KO bone marrow derived stromal cells toward the tumor microenvironment in vivo. WT mice with CD44KO bone marrow had less CD44KOderived tumor stroma compared to mice with WT bone marrow. These results indicate that CD44 is crucial to stromal cell migration and incorporation to the tumor microenvironment as TAF.

REST maintains self-renewal and pluripotency of embryonic stem cells.

The neuronal repressor REST (RE1-silencing transcription factor; also called NRSF) is expressed at high levels in mouse embryonic stem (ES) cells, but its role in these cells is unclear. Here we show that REST maintains self-renewal and pluripotency in mouse ES cells through suppression of the microRNA miR-21. We found that, as with known self-renewal markers, the level of REST expression is much higher in self-renewing mouse ES cells than in differentiating mouse ES (embryoid body, EB) cells. Heterozygous deletion of Rest (Rest+/-) and its short-interfering-RNA-mediated knockdown in mouse ES cells cause a loss of self-renewal-even when these cells are grown under self-renewal conditions-and lead to the expression of markers specific for multiple lineages. Conversely, exogenously added REST maintains self-renewal in mouse EB cells. Furthermore, Rest+/- mouse ES cells cultured under self-renewal conditions express substantially reduced levels of several self-renewal regulators, including Oct4 (also called Pou5f1), Nanog, Sox2 and c-Myc, and exogenously added REST in mouse EB cells maintains the self-renewal phenotypes and expression of these self-renewal regulators. We also show that in mouse ES cells, REST is bound to the gene chromatin of a set of miRNAs that potentially target self-renewal genes. Whereas mouse ES cells and mouse EB cells containing exogenously added REST express lower levels of these miRNAs, EB cells, Rest+/- ES cells and ES cells treated with short interfering RNA targeting Rest express higher levels of these miRNAs. At least one of these REST-regulated miRNAs, miR-21, specifically suppresses the self-renewal of mouse ES cells, corresponding to the decreased expression of Oct4, Nanog, Sox2 and c-Myc. Thus, REST is a newly discovered element of the interconnected regulatory network that maintains the self-renewal and pluripotency of mouse ES cells.

A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells.

Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute approximately 60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the differentiation of hES cells into an essentially pure (>99%) population of ATII cells (hES-ATII). Purity, as well as biological features and morphological characteristics of normal ATII cells, was demonstrated for the hES-ATII cells, including lamellar body formation, expression of surfactant proteins A, B, and C, alpha-1-antitrypsin, and the cystic fibrosis transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5. Collectively, these data document the successful generation of a pure population of ATII cells derived from hES cells, providing a practical source of ATII cells to explore in disease models their potential in the regeneration and repair of the injured alveolus and in the therapeutic treatment of genetic diseases affecting the lung.

Expression of the neural stem cell markers NG2 and L1 in human angiomyolipoma: are angiomyolipomas neoplasms of stem cells?

Angiomyolipomas are benign tumors of the kidney which express phenotypes of smooth muscle, fat, and melanocytes. These tumors appear with increased frequency in the autosomal dominant disorder tuberous sclerosis and are the leading cause of morbidity in adults with tuberous sclerosis. While benign, these tumors are capable of provoking life threatening hemorrhage and replacement of the kidney parenchyma, resulting in renal failure. The histogenesis of these tumors is currently unclear, although currently, we believe these tumors arise from "perivascular epithelioid cells" of which no normal counterpart has been convincingly demonstrated. Recently, stem cell precursors have been recognized that can give rise to smooth muscle and melanocytes. These precursors have been shown to express the neural stem cell marker NG2 and L1. In order to determine whether angiomyolipomas, which exhibit smooth muscle and melanocytic phenotypes, express NG2 and L1, we performed immunocytochemistry on a cell line derived from a human angiomyolipoma, and found that these cells are uniformly positive. Immunohistochemistry of human angiomyolipoma specimens revealed uniform staining of tumor cells, while renal cell carcinomas revealed positivity only of angiogenic vessels. These results support a novel histogenesis of angiomyolipoma as a defect in differentiation of stem cell precursors.

LMW-E MEDIATES MAMMARY TUMORIGENESIS BY DEREGULATING ACINAR MORPHOGENESIS & GENERATING CANCER STEM CELLS

Cyclin E is the regulatory subunit of the cyclin E/CDK2 complex that mediates the G1-S phase transition. N-terminal cleavage of cyclin E by elastase in breast cancer generates two low molecular weight (LMW) isoforms that exhibit both enhanced kinase activity and resistance to p21 and p27 inhibition compared to fulllength cyclin E. Clinically, approximately 27% of breast cancer patients overexpress LMW-E and associate with poor survival. Therefore, we hypothesize that LMW-E disrupts normal mammary acinar morphogenesis and serves as the initial route into breast tumor development. We first demonstrate that LMW-E overexpression in non-tumorigenic hMECs is sufficient to induce tumor formation in athymic mice significantly more than overexpression of full-length cyclin E and requires CDK2- associated kinase activity. Further in vivo passaging of these tumors augments LMW-E expression and tumorigenic potential. When subjected to acinar morphogenesis in vitro, LMW-E mediates significant morphological disruption by generating hyperproliferative and multi-acinar complexes. Proteomic analysis of patient tissues and tumor cells with high LMW-E expression reveals that the activation of the b-Raf-ERK1/2-mTOR pathway in concert with high LMW-E expression predicts poor patient survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (b-raf inhibitor) effectively prevented aberrant acinar formation in LMW-E-expressing cells by inducing the G1/S cell cycle arrest. In addition, the LMW-E-expressing tumor cells exhibit phenotypes characteristic of the EMT and enhanced cellular invasiveness. These tumor cells also enrich for cells with CSC phenotypes such as increased CD44hi/CD24lo population, enhanced mammosphere formation, and upregulation of ALDH expression and enzymatic activity. Furthermore, the CD44hi/CD24lo population also shows positive correlation with LMW-E expression in both the tumor cell line model and breast cancer patient samples (p<0.0001 & p=0.0435, respectively). Combination treatment using doxorubicin and salinomycin demonstrates synergistic cytotoxic effects in cells with LMW-E expression but not in those with full-length cyclin E expression. Finally, ProtoArray microarray identifies Hbo1 as a novel substrate of the cyclin E/CDK2 complex and its overexpression results in enrichment for CSCs. Collectively, these data emphasize the strong oncogenic potential of LMW-E in mammary tumorigenesis and suggest possible therapeutic strategies to treat breast cancer patients with high LMW-E expression.

DNA damage-induced Fas ligand expression by epidermal dendritic cells mediates UV-induced immune suppression of contact hypersensitivity

Exposure to UVB radiation induces local and systemic immune suppression, evidenced by inhibition of the contact hypersensitivity response (CHS). Epidermal dendritic cells, the primary antigen presenting cells responsible for the induction of CHS, are profoundly altered in phenotype and function by UVB exposure and possess UV-specific DNA damage upon migrating to skin-draining lymph nodes. Expression of the proapoptotic protein FasL has been demonstrated in both skin and lymph node cells following UVB exposure. Additionally, functional FasL expression has recently been demonstrated to be required in the phenomenon of UV-induced immune suppression. To test the hypothesis that FasL expression by DNA-damaged Langerhans cells migrating to the skin-draining lymph nodes is a crucial event in the generation of this phenomenon, mice were given a single 5KJ/m2 UV-B exposure and sensitized to 0.5% FITC through the exposed area. Dendritic cells (DC) harvested from skin-draining lymph nodes (DLN) 18 hours following sensitization by magnetic CD11c-conjugated microbeads expressed high levels of Iab, CD80 and CD86, DEC-205 and bore the FITC hapten, suggesting epidermal origin. Radioimmunoassay of UV-specific DNA damage showed that DC contained the vast majority of cyclobutane pyrimidine dimers (CPDs) found in the DLN after UVB and exhibited increased FasL mRNA expression, a result which correlated with greatly increased FasL-mediated cytotoxicity. The ability of DCs to transfer sensitization to naïve hosts was lost following UVB exposure, a phenomenon which required DC FasL expression, and was completely reversed by cutaneous DNA repair. Collectively, these results demonstrate the central importance of DNA damage-induced FasL expression on migrating dendritic cells in mediating UV-induced suppression of contact hypersensitivity. ^

Examination of the engraftment of exogenous mesenchymal stem cells in ovarian tumors and their potential use as delivery vehicles for therapeutic genes

Interactions between neoplastic cells and the host stroma play a role in both tumor cell migration and proliferation. Stromal cells provide structural support for malignant cells, modulate the tumor microenvironment, and influence phenotypic behavior as well as the aggressiveness of the malignancy. In response, the tumor provides growth factors, cytokines, and cellular signals that continually initiate new stromal reactions and recruit new cells into the microenvironment to further support tumor growth. Since growing tumors recruit local cells, as well as supplemental cells from the circulation, such as fibroblasts and endothelial precursors, the question arises if it would be possible to access circulating stromal cells to modify the tumor microenvironment for therapeutic benefits. One such cell type, mesenchymal stem cells (MSC), could theoretically be engrafted into stroma. MSC are pluripotent cells that have been shown to form stromal elements such as myofibroblasts, perivascular tissues and connective tissues. Several reports have demonstrated that MSC can incorporate into sites of wound healing and tissue repair, due to active tissue remodeling and local paracrine factors, and given the similarity between wound healing and the carcinoma induced stromal response one can hypothesize that MSC have the potential to be recruited to sites of tumor development. In addition, gene-modified MSC could be used as cellular vehicles to deliver gene products into tumors. My results indicate that MSC home to and participate in tumor stroma formation in ovarian tumor xenografts in mice. Additionally, once homed to tumor beds, MSC proliferate rapidly and integrate. My studies aim at understanding the fate of MSC in the tumor microenvironment, as well as utilizing them for cellular delivery of therapeutic genes into the stroma of ovarian carcinomas. ^

LOW REACTIVE OXYGEN SPECIES AND HIGH GLYCOLYSIS IN GLIOBLASTOMA STEM CELLS:MECHANISMS AND THERAPEUTIC IMPLICATIONS

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor with poor prognosis due in part to drug resistance and high incidence of tumor recurrence. The drug resistant and cancer recurrence phenotype may be ascribed to the presence of glioblastoma stem cells (GSCs), which seem to reside in special stem-cell niches in vivo and require special culture conditions including certain growth factors and serum-free medium to maintain their stemness in vitro. Exposure of GSCs to fetal bovine serum (FBS) can cause their differentiation, the underlying mechanism of which remains unknown. Reactive oxygen species (ROS) play an important role in normal stem cell differentiation, but their role in affecting cancer stem cell fate remains unclear. Whether the metabolic characteristics of GSCs are different from other glioblastoma cells and can be targeted are also unknown. In this study, we used several stem-like glioblastoma cell lines derived from clinical tissues by typical neurosphere culture system or orthotopic xenografts, and showed that addition of fetal bovine serum to the medium induced an increase of ROS, leading to aberrant differentiation and decreases of stem cell markers such as CD133. We found that exposure of GSCs to serum induced their differentiation through activation of mitochondrial respiration, leading to an increase in superoxide (O2-) generation and a profound ROS stress response manifested by upregulation of oxidative stress response pathway. This increase in mitochondrial ROS led to a down-regulation of molecules including SOX2, and Olig2, and Notch1 that are important for stem cell function and an upregulation of mitochondrial superoxide dismutase SOD2 that converts O2- to H2O2. Neutralization of ROS by antioxidant N-acetyl-cysteine in the serum-treated GSCs suppressed the increase of superoxide and partially rescued the expression of SOX2, Olig2, and Notch1, and prevented the serum-induced differentiation phenotype. Additionally, GSCs showed high dependence on glycolysis for energy production. The combination of a glycolytic inhibitor 3-BrOP and a chemotherapeutic agent BCNU depleted cellular ATP and inhibited the repair of BCNU-induced DNA damage, achieving strikingly synergistic killing effects in drug resistant GSCs. This study uncovers the metabolic properties of glioblastoma stem cells and suggests that mitochondrial function and cellular redox status may profoundly affect the fates of glioblastoma stem cells via a ROS-mediated mechanism, and that the active glycolytic metabolism in cancer stem cells may provide a biochemical basis for developing novel therapeutic strategies to effectively eliminate GSCs.

Potential REST Targets in Neural Stem Cells and Glioblastoma-Derived Stem Cells

Glioblastoma multiforme (GBM) tumors are the most common malignant primary brain tumors in adults. The current theory is that these tumors are caused by self-renewing glioblastoma-derived stem cells (GSCs). At the current time, the mechanisms that regulate self-renewal and other oncogenic properties of GSCs remain unknown. Recently, we found transcriptional repressor REST maintains self-renewal in neural stem cells (NSCs) and in GSCs. REST also regulates other oncogenic properties, such as apoptosis, invasion and proliferation. However, the mechanisms by which REST regulates these oncogenic properties are unknown. In an attempt to determine these mechanisms, we performed loss and gain-of-function experiments and genome-wide mRNA expression analysis in GSCs, and we were able to identify REST-regulated genes in GSCs. This was accomplished, after screening concordantly regulated genes in NSCs and GSCs, utilizing two RE1 databases, and setting two-fold expression as filters on the resulting genes. These results received further validation by qRT-PCR. Ingenuity Pathway Analysis (IPA) analysis further revealed the top REST target genes in GSCs were downstream targets of REST and/or involved in other cancers in other cell lines. IPA also revealed that many of the differentially-regulated genes identified in this study are involved in oncogenic properties seen in GBM, and which we believe are related to REST expression.

PROSTATE CANCER STEM CELLS: DRUG TOLERANCE, EXPERIMENTAL RECONSTITUTION AND CASTRATION RESISTANCE

Prostate cancer (PCa) is one of the leading malignancies affecting men in the Western world. Although tremendous effort has been made towards understanding PCa development and developing clinical treatments in the past decades, the exact mechanisms of PCa are still not clearly understood. Emerging evidence has postulated that a population of stem cell-like cells inside a tumor, termed ‘cancer stem cells (CSCs)’, may be the cells responsible for tumor initiation, progression, recurrence, metastasis and therapy resistance. Like CSC studies in other cancer types, it has been reported that PCa also contains CSCs. However, there remain several unresolved questions that need to be clarified. First, the relationship between prostate CSCs (PCSCs) and therapy resistance (chemo- and radio-) is not known. Herein, we have found that not all CSCs are drug-tolerant, and not all drug-tolerant cells are CSCs. Second, whether primary human PCa (HPCa) actually contain PCSCs remains unclear, due to the well-known fact that we have yet to establish a reliable assay system that can reproducibly and faithfully reconstitute tumor regeneration from single HPCa cells. Herein, after utilizing more than 114 HPCa samples we have provided evidence that immortalized bone marrow-derived stromal cells (Hs5) can help dissociated HPCa cells generate undifferentiated tumors in immunodeficient NOD/SCID-IL2Rγ-/- mice, and the undifferentiated PCa cells seem to have a survival advantage to generate tumors. Third, the evolution of PCa from androgen dependent to the lethally castration resistant (CRPC) stage remains enigmatic, and the cells responsible for CRPC development have not been identified. Herein, we have found a putative cell population, ALDH+CD44+α2β1+ PCa cells that may represent a cell-of-origin for CRPC. Taken together, our work has improved our understanding of PCSC properties, possibly highlighting a potential therapeutic target for CRPC.

GENOME-WIDE PROFILING UNVEILS CRITICIAL FUNCTIONS OF p53 IN HUMAN EMBRYONIC STEM CELLS

Embryonic stem cells (ESCs) possess two unique characteristics: infinite self-renewal and the potential to differentiate into almost every cell type (pluripotency). Recently, global expression analyses of metastatic breast and lung cancers revealed an ESC-like expression program or signature, specifically for cancers that are mutant for p53 function. Surprisingly, although p53 is widely recognized as the guardian of the genome, due to its roles in cell cycle checkpoints, programmed cell death or senescence, relatively little is known about p53 functions in normal cells, especially in ESCs. My hypothesis is that p53 has specific transcription regulatory functions in human ESCs (hESCs) that a) oppose pluripotency and b) protect the stem cell genome in response to DNA damage and stress signaling. In mouse ESCs, these roles are believed to coincide, as p53 promotes differentiation in response to DNA damage, but this is unexplored in hESCs. To determine the biological roles of p53, specifically in hESCs, we mapped genome-wide chromatin interactions of p53 by chromatin immunoprecipitation and massively parallel tag sequencing (ChIP-Seq), and did so under three VIdifferent conditions of hESC status: pluripotency, differentiation-initiated and DNA-damage-induced. ChIP-Seq showed that p53 is enriched at distinct, induction-specific gene loci during each of these different conditions. Microarray gene expression analysis and functional annotation of the distinct p53-target genes revealed that p53 regulates specific genes encoding developmental regulators, which are expressed in differentiation-initiated but not DNA- damaged hESCs. We further discovered that, in response to differentiation signaling, p53 binds regions of chromatin that are repressed but also poised for rapid activation by core pluripotency factors OCT4 and NANOG in pluripotent hESCs. In response to DNA damage, genes associated with migration and motility are targeted by p53; whereas, the prime targets of p53 in control of cell death are conserved for p53 regulation in both differentiation and DNA damage. Our genome-wide profiling and bioinformatics analyses show that p53 occupies a special set of developmental regulatory genes during early differentiation of hESCs and functions in an induction-specific manner. In conclusion, our research unveiled previously unknown functions of p53 in ESC biology, which augments our understanding of one of the most deregulated proteins in human cancers.

Use of Echogenic Immunoliposomes for Delivery of both Drug and Stem Cells for Inhibition of Atheroma Progression

Use of Echogenic Immunoliposomes for Delivery of both Drug and Stem Cells for Inhibition of Atheroma Progression By Ali K. Naji B.S. Advisor: Dr. Melvin E. Klegerman PhD Background and significance: Echogenic liposomes can be used as drug and cell delivery vehicles that reduce atheroma progression. Vascular endothelial growth factor (VEGF) is a signal protein that induces vasculogenesis and angiogenesis. VEGF functionally induces migration and proliferation of endothelial cells and increases intracellular vascular permeability. VEGF activates angiogenic transduction factors through VEGF tyrosine kinase domains in high-affinity receptors of endothelial cells. Bevacizumab is a humanized monoclonal antibody specific for VEGF-A which was developed as an anti-tumor agent. Often, anti-VEGF agents result in regression of existing microvessels, inhibiting tumor growth and possibly causing tumor shrinkage with time. During atheroma progression neovasculation in the arterial adventitia is mediated by VEGF. Therefore, bevacizumab may be effective in inhibiting atheroma progression. Stem cells show an ability to inhibit atheroma progression. We have previously demonstrated that monocyte derived CD-34+ stem cells that can be delivered to atheroma by bifunctional-ELIP ( BF-ELIP) targeted to Intercellular Adhesion Molecule-1 (ICAM-1) and CD-34. Adhesion molecules such as ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) are expressed by endothelial cells under inflammatory conditions. Ultrasound enhanced liposomal targeting provides a method for stem cell delivery into atheroma and encapsulated drug release. This project is designed to examine the ability of echogenic liposomes to deliver bevacizumab and stem cells to inhibit atheroma progression and neovasculation with and without ultrasound in vitro and optimize the ultrasound parameters for delivery of bevacizumab and stem cells to atheroma. V Hypotheses: Previous studies showed that endothelial cell VEGF expression may relate to atherosclerosis progression and atheroma formation in the cardiovascular system. Bevacizumab-loaded ELIP will inhibit endothelial cell VEGF expression in vitro. Bevacizumab activity can be enhanced by pulsed Doppler ultrasound treatment of BEV-ELIP. I will also test the hypothesis that the transwell culture system can serve as an in vitro model for study of US-enhanced targeted delivery of stem cells to atheroma. Monocyte preparations will serve as a source of CD34+ stem cells. Specific Aims: Induce VEGF expression using PKA and PKC activation factors to endothelial cell cultures and use western blot and ELISA techniques to detect the expressed VEGF.  Characterize the relationship between endothelial cell proliferation and VEGF expression to develop a specific EC culture based system to demonstrate BEV-ELIP activity as an anti-VEGF agent. Design a cell-based assay for in vitro assessment of ultrasound-enhanced bevacizumab release from echogenic liposomes.  Demonstrate ultrasound delivery enhancement of stem cells by applying different types of liposomes on transwell EC culture using fluorescently labeled monocytes and detect the effect on migration and attachment rate of these echogenic liposomes with and without ultrasound in vitro.

Inducible caspase 9 suicide gene to improve the safety of allodepleted T cells after haploidentical stem cell transplantation.

Addback of donor T cells following T cell-depleted stem cell transplantation (SCT) can accelerate immune reconstitution and be effective against relapsed malignancy. After haploidentical SCT, a high risk of graft-versus-host disease (GVHD) essentially precludes this option, unless the T cells are first depleted of alloreactive precursor cells. Even then, the risks of severe GVHD remain significant. To increase the safety of the approach and thereby permit administration of larger T cell doses, we used a suicide gene, inducible caspase 9 (iCasp9), to transduce allodepleted T cells, permitting their destruction should administration have adverse effects. We made a retroviral vector encoding iCasp9 and a selectable marker (truncated CD19). Even after allodepletion (using anti-CD25 immunotoxin), donor T cells could be efficiently transduced, expanded, and subsequently enriched by CD19 immunomagnetic selection to >90% purity. These engineered cells retained antiviral specificity and functionality, and contained a subset with regulatory phenotype and function. Activating iCasp9 with a small-molecule dimerizer rapidly produced >90% apoptosis. Although transgene expression was downregulated in quiescent T cells, iCasp9 remained an efficient suicide gene, as expression was rapidly upregulated in activated (alloreactive) T cells. We have demonstrated the clinical feasibility of this approach after haploidentical transplantation by scaling up production using clinical grade materials.

ΔNp63 REGULATES A COMPLEX NETWORK OF TARGET GENES IN LIMB AND EPIDERMAL DEVELOPMENT

The skin is composed of two major compartments, the dermis and epidermis. The epidermis forms a barrier to protect the body. The stratified epithelium has self-renewing capacity throughout life, and continuous turnover is mediated by stem cells in the basal layer. p63 is structurally and functionally related to p53. In spite of their structural similarities, p63 is critical for the development and maintenance of stratified epithelial tissues, unlike p53. p63 is highly expressed in the epidermis and previously has been shown to play a critical role in the development and maintenance of the epidermis. The study of p63 has been complicated due to the existence of multiple isoforms: those with a transactivation domain (TAp63) and those lacking this domain (ΔNp63). Mice lacking p63 cannot form skin, have craniofacial and skeletal defects and die within hours after birth. These defects are due to the ability of p63 to regulate multiple processes in skin development including epithelial stem cell proliferation, differentiation, and adherence programs. To determine the roles of these isoforms in skin development and maintenance, isoform specific p63 conditional knock out mice were generated by our lab. TAp63-/- mice age prematurely, develop blisters, and display wound-healing defects that result from hyperproliferation of dermal stem cells. That results in premature depletion of these cells, which are necessary for wound repair, that indicates TAp63 plays a role in dermal/epidermal maintenance. To study the role of ΔNp63, I generated a ΔNp63-/- mouse and analyzed the skin by performing immunofluorescence for markers of epithelial differentiation. The ΔNp63-/- mice developed a thin, disorganized epithelium but differentiation markers were expressed. Interestingly, the epidermis from ΔNp63-/- mice co-expressed K14 and K10 in the same cell suggesting defects in epidermal differentiation and stratification. This phenotype is reminiscent of the DGCR8fl/fl;K14Cre and Dicerfl/fl;K14Cre mice skin. Importantly, DGCR8-/- embryonic stem cells (ESCs) display a hyperproliferation defect by failure to silence pluripotency genes. Furthermore, I have observed that epidermal cells lacking ΔNp63 display a phenotype reminiscent of embryonic stem cells instead of keratinocytes. Thus, I hypothesize that genes involved in maintaining pluripotency, like Oct4, may be upregulated in the absence of ΔNp63. To test this, q-RT PCR was performed for Oct4 mRNA with wild type and ΔNp63-/- 18.5dpc embryo skin. I found that the level of Oct4 was dramatically increased in the absence of ΔNp63-/-. Based on these results, I hypothesized that ΔNp63 induces differentiation by silencing pluripotency regulators, Oct4, Sox2 and Nanog directly through the regulation of DGCR8. I found that DGCR8 restoration resulted in repression of Oct4, Sox2 and Nanog in ΔNp63-/- epidermal cells and rescue differentiation defects. Loss of ΔNp63 resulted in pluripotency that caused defect in proper differentiation and stem cell like phenotype. This led me to culture the ΔNp63-/- epidermal cells in neuronal cell culture media in order to address whether restoration of DGCR8 can transform epidermal cells to neuronal cells. I found that DGCR8 restoration resulted in a change in cell fate. I also found that miR470 and miR145 play a role in the induction of pluripotency by repressing Oct4, Sox2 and Nanog. This indicates that ΔNp63 induces terminal differentiation through the regulation of DGCR8.