AP Cells Research Articles

Expression of the apelin system in the porcine pituitary during the oestrous cycle and early pregnancy and the effect of apelin on LH and FSH secretion

Reproductive success requires considerable energy investment. Research has shown that some adipokines, i.e. the hormones produced in adipose tissue, affect reproductive functions by influencing all structures of the hypothalamic-pituitary-ovarian axis. Apelin is a recently identified member of the adipokine family. To the best of the authors' knowledge, this is the first study to investigate the gene and protein expression of the apelin system (the apelin hormone and the apelin receptor, APJ) in the anterior (AP) and posterior (PP) pituitary lobes of the domestic pig during different phases of the oestrous cycle (days 2 to 3, 10 to 12, 14 to 16, and 17 to 19) and in early pregnancy (days 10 to 11, 12 to 13, 15 to 16, and 27 to 28). It was also assumed that apelin participates in the regulation of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion and influences Akt, MAPK/Erk1/2, and AMPK signalling pathways in the AP during the oestrous cycle. Apelin, APJ mRNAs and proteins were detected in both pituitary lobes. Apelin was identified in gonadotropes, somatotropes, lactotropes, and thyrotropes. The study also revealed that apelin and APJ mRNA/protein levels fluctuate during the oestrous cycle and early gestation. Apelin affects basal, GnRH- and/or insulin-stimulated gonadotropin secretion in some phases of the cycle, as well as the phosphorylation of Akt, MAPK/Erk1/2, and AMPK proteins in AP cells. These findings suggest that apelin may be produced locally in the pituitary and that this gland is receptive to apelin's action. The study also suggest that apelin may influence female reproductive functions by controlling the release of LH and FSH from AP cells, and that it affects Akt, MAPK/Erk1/2, and AMPK signalling pathways.

Visfatin in the porcine pituitary gland: expression and regulation of secretion during the oestrous cycle and early pregnancy

Visfatin is a multifunctional protein which, besides the control of energy homeostasis, seems to be also involved in the regulation of female fertility through the influence on the endocrine hypothalamus-pituitary-gonadal axis, including the pituitary. The aim of this study was to investigate the expression of visfatin mRNA and protein in the anterior (AP) and posterior pituitary lobes of the pig during the oestrous cycle and early pregnancy. In AP, we also examined colocalisation of visfatin with pituitary tropic hormones. Moreover, we aimed to evaluate the in vitro effects of GnRH, FSH, LH, and insulin on visfatin protein concentration and secretion in AP cells during the cycle. The study showed that visfatin is present in all types of porcine pituitary endocrine cells and its expression is reliant on stage of the cycle or pregnancy. GnRH, FSH, LH and insulin stimulated visfatin secretion by AP cells on days 17 to 19 of the cycle, while on days 2 to 3 visfatin release was enhanced only by LH. Summarising, visfatin is locally produced in the pituitary in a way dependent on hormonal milieu typical for reproductive status of pigs. Further research is required to clarify the role of visfatin in the pituitary gland.

Biological control of soft rot in potato by κ-carrageenan carriers encapsulated microbial predators.

The Pectobacterium and Dickeya pectinolytic bacteria are phytopathogens responsible for several macerating diseases on a wide range of crops and ornamental plants. Recently, bacterial predators belonging to the Bdellovibrio and like organisms (BALOs) were shown to efficiently prey on these rot-causing bacteria and reduce soft rot-induced potato slice maceration. In the current research, our novel approach aimed at developing and studying a κ-carrageenan-based encapsulation system for fast-release of entrapped B. bacteriovorus HD100 in high numbers to prevent bacterial soft-rot infections. κ-carrageenan-dried carriers swelled and dissolved upon immersion in water due to a loss of potassium ions which are the main cross-linking agents. Survival rates of the predators after drying were higher for immobilized bdelloplasts (e.g., predator inside the host) compared to attack phase (host-searching, AP) cells, and with the addition of the osmoprotectant trehalose to the carriers. Released encapsulated predators preyed efficiently on soft rot bacteria, with bdelloplasts performing better as compared to AP cells. However, predation dynamics were influenced by the type of added osmoprotectant. Carrageenan-trehalose carriers encapsulating predators were able to reduce soft-rot disease in situ using a potato slice assay. To our knowledge, this research is the first to explore the potential of encapsulated BALOs against phytopathogens. KEY POINTS: • Dissolution of the carriers was affected by potassium concentration in the system. • Encapsulation of bdelloplasts with trehalose best maintained the predator viability. • The encapsulated predators efficiently controlled soft rot in vitro and in situ.

Abstract 5570: Development of piggyBac transposon-mediated HER2-CAR-T cells for the treatment of solid tumors

Abstract Background: Although chimeric antigen receptor (CAR)-T therapies have achieved remarkable success in the treatment of hematologic malignancies, the outcome for patients with solid tumors remains poor. There are several reasons behind this, including exhaustion of CAR-T cell, poor homing and penetration in the tumor, and the lack of persistence in the immunosuppressive tumor microenvironment. To solve these problems, we have developed HER2-CAR-T cells (BP2301) using the piggyBac (PB) transposon-based gene transfer system. Methods and Results: Second generation HER2-CAR construct plus PB transposase were introduced into autologous peripheral blood mononuclear cells (PBMC) by electroporation. These cells were activated with UV-inactivated genetically-engineered antigen presenting autologous PBMC (AP cells) expressing HER2, CD80, and 4-1BBL, and propagated for 14 days to obtain the final product (BP2301). BP2301 exhibited dominant fraction of less exhausted stem cell memory-like T cells (Tscm), which could be associated with antitumor efficacy. BP2301 showed robust in vitro killing activity against HER2+ tumor cells even after multiple rounds of cancer cell challenge, suggesting the long-term functionality without immune-exhaustion. In a xenograft murine model of rhabdomyosarcoma cell line (SJCRh30), a single intravenous administration of BP2301 (5 × 106 CAR-positive cells) eradicated the tumor, and mice treated with BP2301 rejected the second tumor establishment owing to the in vivo expansion of BP2301. Conclusion: BP2301 has demonstrated potent anti-tumor activity in vitro and in vivo. We have established a GMP manufacturing process for BP2301, completed non-clinical safety and efficacy studies, and plan to launch an investigator-initiated phase 1 clinical trial to evaluate the safety and efficacy of BP2301 in HER2-expressing sarcoma and gynecologic malignancies in the second quarter of 2022. Citation Format: Koichiro Shioya, Tomio Matsumura, Yuta Ohira, Naomi Komatsuzaki, Manaka Shinagawa, Miyuki Tanaka, Shigeki Yagyu, Yozo Nakazawa, Lilin Zhang. Development of piggyBac transposon-mediated HER2-CAR-T cells for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5570.

Single-Cell RNA-Seq Analysis Reveals Lung Epithelial Cell Type-Specific Responses to HDM and Regulation by Tet1.

Tet1 protects against house dust mite (HDM)-induced lung inflammation in mice and alters the lung methylome and transcriptome. In order to explore the role of Tet1 in individual lung epithelial cell types in HDM-induced inflammation, we established a model of HDM-induced lung inflammation in Tet1 knockout and littermate wild-type mice, then studied EpCAM+ lung epithelial cells using single-cell RNA-seq analysis. We identified eight EpCAM+ lung epithelial cell types, among which AT2 cells were the most abundant. HDM challenge altered the relative abundance of epithelial cell types and resulted in cell type-specific transcriptomic changes. Bulk and cell type-specific analysis also showed that loss of Tet1 led to the altered expression of genes linked to augmented HDM-induced lung inflammation, including alarms, detoxification enzymes, oxidative stress response genes, and tissue repair genes. The transcriptomic regulation was accompanied by alterations in TF activities. Trajectory analysis supports that HDM may enhance the differentiation of AP and BAS cells into AT2 cells, independent of Tet1. Collectively, our data showed that lung epithelial cells had common and unique transcriptomic signatures of allergic lung inflammation. Tet1 deletion altered transcriptomic networks in various lung epithelial cells, which may promote allergen-induced lung inflammation.

Malignant subclone drives metastasis of genetically and phenotypically heterogenous cell clusters through fibrotic niche generation

A concept of polyclonal metastasis has recently been proposed, wherein tumor cell clusters break off from the primary site and are disseminated. However, the involvement of driver mutations in such polyclonal mechanism is not fully understood. Here, we show that non-metastatic AP cells metastasize to the liver with metastatic AKTP cells after co-transplantation to the spleen. Furthermore, AKTP cell depletion after the development of metastases results in the continuous proliferation of the remaining AP cells, indicating a role of AKTP cells in the early step of polyclonal metastasis. Importantly, AKTP cells, but not AP cells, induce fibrotic niche generation when arrested in the sinusoid, and such fibrotic microenvironment promotes the colonization of AP cells. These results indicate that non-metastatic cells can metastasize via the polyclonal metastasis mechanism using the fibrotic niche induced by malignant cells. Thus, targeting the fibrotic niche is an effective strategy for halting polyclonal metastasis.

Relative abundance of chemerin mRNA transcript and protein in pituitaries of pigs during the estrous cycle and early pregnancy and associations with LH and FSH secretion during the estrous cycle

Adipokines such as chemerin affect metabolic status and reproductive function in many species. The hypothesis in the present study was that there were chemerin mRNA transcript and protein in the pituitary of pigs and that relative abundances fluctuate during the estrous cycle and early pregnancy. Chemerin is thought to modulate luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion during the estrous cycle. Changes in the relative abundance of chemerin mRNA transcript and protein in anterior (AP) and posterior (PP) pituitaries of pigs were investigated, for the first time in the present study, during four phases of the estrous cycle and four periods of early pregnancy. Chemerin protein was localized in gonadotrophs, thyrotrophs and somatotrophs during the estrous cycle and early gestation. Chemerin treatments affected both basal, GnRH- and/or insulin-induced LH and FSH production, with there being variations with phase of the estrous cycle when tissues were collected. These findings indicate chemerin may be produced locally in the pituitary and may affect female reproductive function by controlling the release of LH and FSH from AP cells.

Abstract 969: Characterization of murine mammary epithelial populations throughout development and in premalignant tissue via mass cytometry

Abstract Single-cell technologies provide a unique opportunity to develop a comprehensive understanding of the dynamic transitions of mammary epithelial cells during normal development, pregnancy, and tumorigenesis. To provide a higher resolution map of epithelial cell dynamics, we developed a mass cytometry panel composed of 39 antibodies targeting cell surface antigens of distinct murine mammary populations. We first utilized this panel to evaluate mammary epithelial composition through puberty and virgin adulthood. This analysis identified subpopulations of the three major mammary epithelial lineages (basal, alveolar progenitor [AP], and hormone-sensing cells [HS]). Interestingly, no clusters expressed markers consistent with their being intermediate between basal and luminal populations or the two luminal populations. We also identified puberty-restricted populations in each of the three lineages. Intriguingly, the luminal pubertal populations expressed basal markers such as CD104 and CD73. We also examined epithelial dynamics during a pregnancy cycle, including mid-pregnancy, lactation, involution, and post-involution. Three hitherto undescribed transit-amplifying clusters were enriched during pregnancy and persisted through involution. These clusters were highly proliferative (as measured by Idu incorporation) and expressed several basal markers, whose expression persisted among AP cells during involution and among a subset of post-involution AP cells. Finally, we analyzed alterations in mammary tissue in the context of loss of tumor suppressors (Apcmut/+, Atm+/-, Cdkn1a-/-, Arf-/-, Trp53+/-, or Trp53-/-) or expression of oncogenes (MMTV-Errb2, -neu, or -PyMT). Myriad alterations were observed across the conditions, the most striking of which was restriction of alterations in MMTV models to cells in the AP lineage. The expression of PyMT increased the frequency and basal-like features of AP cells. Analysis of a PyMT-induced tumor also showed that tumor cells were overwhelming of an AP-like phenotype. In conclusion, our data paint a high-resolution portrait of mammary epithelial dynamics throughout development and in the context of pro-tumorigenic genetic alterations. We report numerous novel populations during puberty and the pregnancy cycle. A particularly intriguing observation is that luminal populations appear to acquire basal-like differentiation features during puberty, pregnancy, and pre-tumorigenesis, suggesting the oft-reported mixed luminal/basal differentiation status of breast cancer could be a coopting of normal developmental programs. This result may provide clues for uncovering and blocking the molecular mechanisms of breast carcinogenesis. Citation Format: Gary K. Gray, Joan S. Brugge. Characterization of murine mammary epithelial populations throughout development and in premalignant tissue via mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 969.

S100A4: a novel partner for heat shock protein 47 in antler stem cells and insight into the calcium ion-induced conformational changes

S100A4 is a multiple-function protein highly expressed in tumor or stem cells. We found S100A4 was a novel protein partner for heat shock protein 47 (HSP47) in deer antlerogenic periosteum cells (AP cells), indicating that S100A4 could bind with HSP47. S100A4 had both calcium-dependent and calcium-independent patterns (labeled as SCd and SCi, respectively) to execute different biological activities. Homology models of HSP47, SCd and SCi were constructed. HSP47:collagen model, HSP47:collagen I-V, HSP47:SCd and HSP47:SCi complexes were built using ZDOCK software. Together with free SCd and SCi, 200 ns molecular dynamic (MD) simulations were performed to analyze binding free energies and SCi/SCd conformational changes. The energetic results showed that SCi had the strongest affinity to HSP47, and followed by collagens. SCd had little interaction with HSP47. Decomposition energy results showed that collagen model interacted with HSP47 mainly though neutral amino acids. When SCi bound with HSP47, the majority of mediated amino acids were charged. These results indicated that SCi could compete with collagen on the binding site of HSP47. Root mean square fluctuation (RMSF) values and cross-correlation matrices of principal component analysis (PCA) were calculated to evaluate the SCi/SCd structural variation during MD simulation. Both HSP47 and Ca2+ could stabilize the conformation of SCi/SCd. The loops interacting with Ca2+s and linking the two EF-hand motifs were impacted particularly. The relative moving directions of α-helices in EF-hands were distinct by the binding effect of HSP47 and Ca2+. We found that SCi may regulate the differentiation of AP cells by disturbing the interaction between HSP47 and collagen. Communicated by Ramaswamy H. Sarma

A versatile instrument for ambient pressure x-ray photoelectron spectroscopy: The Lund cell approach

During the past one and a half decades ambient pressure x-ray photoelectron spectroscopy (APXPS) has grown to become a mature technique for the real-time investigation of both solid and liquid surfaces in the presence of a gas or vapour phase. APXPS has been or is being implemented at most major synchrotron radiation facilities and in quite a large number of home laboratories. While most APXPS instruments operate using a standard vacuum chamber as the sample environment, more recently new instruments have been developed which focus on the possibility of custom-designed sample environments with exchangeable ambient pressure cells (AP cells). A particular kind of AP cell solution has been driven by the development of the APXPS instrument for the SPECIES beamline of the MAX IV Laboratory: the solution makes use of a moveable AP cell which for APXPS measurements is docked to the electron energy analyser inside the ultrahigh vacuum instrument. Only the inner volume of the AP cell is filled with gas, while the surrounding vacuum chamber remains under vacuum conditions. The design enables the direct connection of UHV experiments to APXPS experiments, and the swift exchange of AP cells allows different custom-designed sample environments. Moreover, the AP cell design allows the gas-filled inner volume to remain small, which is highly beneficial for experiments in which fast gas exchange is required. Here we report on the design of several AP cells and use a number of cases to exemplify the utility of our approach.

Adiponectin expression in the porcine pituitary during the estrous cycle and its effect on LH and FSH secretion.

Female reproductive success is closely associated with nutritional status and energy balance. In this context, adiponectin appears to be a key hormone connecting reproductive system function and metabolism regulation. It is hypothesized that adiponectin expression in the pituitary depends on the phase of the estrous cycle. The effect of adiponectin on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion is also postulated. Changes in the adiponectin gene and protein expression in the porcine anterior (AP) and posterior (NP) pituitaries as well as the effect of in vitro administration of adiponectin on basal and gonadotropin-releasing hormone (GnRH)- and/or insulin-stimulated LH and FSH secretion were investigated on days 2-3, 10-12, 14-16, and 17-19 of the estrous cycle. Adiponectin gene was more pronounced on days 2-3 in AP but on days 10-12 in NP. Protein concentration in AP was the highest on days 10-12 and in NP on days 10-12 and 17-19 of the cycle. In vitro, adiponectin did not affect basal LH secretion but increased FSH release by AP cells. Adiponectin administration affected GnRH- and/or insulin-induced LH and FSH output in a manner dependent on the phase of the estrous cycle. In this study we indicated for the first time adiponectin expression in the porcine AP and NP that was dependent on the phase of the estrous cycle. In vitro studies indicated that adiponectin may affect gonadotropin secretion. The above suggests that the studied adipokine may influence female reproductive functions via its effect on LH and FSH secretion by gonadotrophs, but the cellular mechanism of its action remains unknown.

Development and characterization of an antibody-labeled super-paramagnetic iron oxide contrast agent targeting prostate cancer cells for magnetic resonance imaging.

In this study we developed, characterized and validated in vitro a functional superparagmagnetic iron-oxide based magnetic resonance contrast agent by conjugating a commercially available iron oxide nanoparticle, Molday ION Rhodamine-B Carboxyl (MIRB), with a deimmunized mouse monoclonal antibody (muJ591) targeting prostate-specific membrane antigen (PSMA). This functional contrast agent is intended for the specific and non-invasive detection of prostate cancer cells that are PSMA positive, a marker implicated in prostate tumor progression and metastasis. The two-step carbodiimide reaction used to conjugate the antibody to the nanoparticle was efficient and we obtained an elemental iron content of 1958±611 per antibody. Immunofluorescence microscopy and flow cytometry showed that the conjugated muJ591:MIRB complex specifically binds to PSMA-positive (LNCaP) cells. The muJ591:MIRB complex reduced cell adhesion and cell proliferation on LNCaP cells and caused apoptosis as tested by Annexin V assay, suggesting anti-tumorigenic characteristics. Measurements of the T2 relaxation time of the muJ591:MIRB complex using a 400 MHz Innova NMR and a multi-echo spin-echo sequence on a 3T MRI (Achieva, Philips) showed a significant T2 relaxation time reduction for the muJ591:MIRB complex, with a reduced T2 relaxation time as a function of the iron concentration. PSMA-positive cells treated with muJ591:MIRB showed a significantly shorter T2 relaxation time as obtained using a 3T MRI scanner. The reduction in T2 relaxation time for muJ591:MIRB, combined with its specificity against PSMA+LNCaP cells, suggest its potential as a biologically-specific MR contrast agent.

Expression of Aquaporins in the Rat Choroid Plexus

Background: Aquaporins (AQPs) are water channels that play a key role in water transport in many epithelia. Recent studies have shown that AQP1, located in the apical membrane of choroid plexus cells, has an important role in cerebrospinal fluid (CSF) production. However, the mechanism of water transport through the basolateral membrane of choroid plexus (CP) cells is yet to be determined. Objectives: In this study, the expression and localization of AQP1, AQP4 and AQP5 in the rat lateral ventricle CP were investigated by immunocytochemistry, western analysis and flowcytometry. Materials and Methods: Choroidal epithelial cells of the lateral ventricle in Wistar rats were isolated and grown in Dulbecco's Modified Eagle's medium <b>(</b>DMEM) supplement, which was refreshed every two days. After five days, the expression patterns of AQP1, AQP4 and AQP5 were determined by immunoblotting, immuncytochemistry and flowcytometry. Results: The immunocytochemistry data revealed the expression of AQP1 and AQP4 in the membrane and the cytoplasmof AP cells, respectively. Through western analysis, the AQP1 antibody detected two bands of approximately 27 and 32 kDa. A single peptide of 29 kDa was recognized by the AQP4 antibody. Quantitative flowcytometry revealed CP cells exhibiting a high level of AQP1 and AQP4 proteins (95.39% and 92.21%, respectively). According to immunocytochemistry, AQP5 is weakly expressed in the cytoplasm of CP cells. Anti-AQP5 antibody recognized a pale band of approximately28 kDa by western analysis. Conclusions: These observations suggested that AQP4 may have an important role in CSF secretion; however, expression of AQP4 or AQP5 was not detected in the cell membrane. Thus, how water crosses the basolateral membrane of CP cells remains to be determined.

Gap Junction-Dependent Homolog Avoidance in the Developing CNS

Oppositely directed projections of some homologous neurons in the developing CNS of the medicinal leech (Hirudo verbana), such as the AP cells, undergo a form of contact-dependent homolog avoidance. Embryonic APs extend axons within the connective nerve toward adjacent ganglia, in which they meet and form gap junctions (GJs) with the oppositely directed axons of their segmental homologs, stop growing, and are later permanently retracted (Wolszon et al., 1994a,b). However, early deletion of an AP neuron leads to resumed growth and permanent maintenance of the projections of neighboring APs. Here we test the hypothesis that a GJ-based signaling mechanism is responsible for this instance of homolog avoidance. We demonstrate that selective knockdown of GJ gene Hve-inx1 expression in single embryonic APs, by expressing a short-hairpin interfering RNA, leads to continued growth of the projections of the cell toward, into, and beyond adjacent ganglia. Moreover, the projections of the APs in adjacent ganglia also resume growth, mimicking their responses to cell deletion. Continued growth was also observed when two different INX1 mutant transgenes that abolish dye coupling between APs were expressed. These include a mutant transgene that effectively downregulates all GJ plaques that include the INX1 protein and a closed channel INX1 mutant that retains the adhesive cellular binding characteristic of INX1 GJs but not the open channel pore function. Our results add GJ intercellular communication to the list of molecular signaling mechanisms that can act as mediators of growth-inhibiting cell-cell interactions that define the topography of neuronal arbors.

Resistance exercise induces region-specific adaptations in anterior pituitary gland structure and function in rats

The anterior pituitary gland (AP) increases growth hormone (GH) secretion in response to resistance exercise (RE), but the nature of AP adaptations to RE is unknown. To that end, we examined the effects of RE on regional AP somatotroph GH release, structure, and relative quantity. Thirty-six Sprague-Dawley rats were assigned to one of four groups: 1) no training or acute exercise (NT-NEX); 2) no training with acute exercise (NT-EX); 3) resistance training without acute exercise (RT-NEX); 4) resistance training with acute exercise (RT-EX). RE incorporated 10, 1 m-weighted ladder climbs at an 85° angle. RT groups trained 3 days/wk for 7 wk, progressively. After death, trunk blood was collected, and each AP was divided into quadrants (ventral-dorsal and left-right). We measured: 1) trunk plasma GH; 2) somatotroph GH release; 3) somatotroph size; 4) somatotroph secretory content; and 5) percent of AP cells identified as somatotrophs. Trunk GH differed by group (NT-NEX, 8.9 ± 2.4 μg/l; RT-NEX, 9.2 ± 3.5 μg/l; NT-EX, 15.6 ± 3.4 μg/l; RT-EX, 23.4 ± 4.6 μg/l). RT-EX demonstrated greater somatotroph GH release than all other groups, predominantly in ventral regions (P < 0.05-0.10). Ventral somatotrophs were larger in NT-EX and RT-NEX compared with RT-EX (P < 0.05-0.10). RT-NEX exhibited significantly greater secretory granule content than all other groups but in the ventral-right region only (P < 0.05-0.10). Our findings indicate reproducible patterns of spatially distinct, functionally different somatotroph subpopulations in the rat pituitary gland. RE training appears to induce dynamic adaptations in somatotroph structure and function.

Abstract 396: Reovirus successfully purges multiple myeloma ex vivo and does not affect human CD34+ cell engraftment in a murine transplantation model.

Abstract Introduction: Multiple Myeloma (MM) is an incurable cancer that accounts for 10% of hematological malignancies. Stem cell rescue following high-dose chemotherapy with autologous or allogenic transplantation has become standard therapy for a subset of good performance patients. Previously we have shown that reovirus, a double stranded RNA virus can successfully purge the RPMI 8226 MM cell line, as well as a patient tumor specimen with no effect on stem cells (Thirukkumaran et al. 2004 : Blood. 102:377-387). In the present study we demonstrate 1) Efficient reovirus ex vivo purging of MM in a murine model of transplantation. 2) Reovirus purging does not affect human CD34+ stem cell repopulation or lineage differentiation. Methods: RPMI 8226-GFP MM cell line was admixed with human apheresis cells (AP) to represent 1% tumor burden. Admixed products were purged with live (LV) or UV-inactivated (DV) reovirus at a multiplicity of infection (MOI) of 40 for 72 hours. To assess purging efficacy and stem cell repopulating ability, SCID/NOD mice were sublethally irradiated and intraperitoneally injected with anti-asialo antibody to eradicate NK cells. Mice were injected intravenously with either live or dead virus purged admixed apheresis cells. Mice showing symptoms of disease were sacrificed and subjected to whole body fluorescence imaging using a GFP Imaging System. Bone marrow cells were collected from mouse femurs and analyzed for CD38+/138+/GFP+ MM cells. Human CD34+ stem cells were isolated from AP cells using Robosep (Stem Cell technologies) and exposed to LV or DV for 72 hours. LV (n=8) and DV (n=5) virus purged stem cells were mixed with sublethally irradiated (2Gy) AP cells (5X 105) and injected into irradiated SCID/NOD mice. Mouse blood and bone marrow was harvested at days 25 or 35 and assessed for human stem cells (CD34+/ 45+), T cells (45+/3+), B cells (19+/45+), NK cells(45+/3-/16+/33+) and myeloid (45+/13+/33+) cells. Results: 100% of LV purged animals (unlike DV purged) were devoid of MM and survived until being sacrificed arbitrarily at day 110 (Kaplan-Meier analysis: p&amp;lt; 0.0012). Both LV and DV purged human stem cells showed re-population in mice and lineage differentiation illustrating a lack of viral effect. Conclusions: The sensitivity of reovirus towards MM and its lack of effect on human stem cells highlight the potential of reovirus as an ex vivo purging agent during autologous hematopoietic stem cell transplants for MM. Currently, we are in the process of studying reovirus purging efficacy of ex vivo MM patient tumor in order to generate data for a future phase I clinical trial. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 396.

Abstract 4935: Ex vivo Acidic Preconditioning Modulates Bone Marrow-derived ckit + Cell Function in vitro and Enhances Their Therapeutic Potential in a Mouse Model of Hindlimb Ischemia

Background and Objective . Bone marrow-derived cell transplantation may provide a novel strategy for the treatment of cardiac and limb ischemia. However, in some cases cells appear to have lost their therapeutic potential and there is an interest in identifying interventions that are clinically applicable and may enhance cells’ ability to repair damaged tissues. The objective of the present work was to examine whether ex vivo acidic preconditioning (AP) may modulate bone marrow-derived ckit + cell functions in vitro and their regenerative properties in a mouse model of hindlimb ischemia. Methods and results. ckit + cells were obtained from 4-week old mice; AP was achieved by exposing cells to hypercarbic acidosis (buffer pH 7.0) for 24 hours and subsequently returning them to pH 7.4. Control ckit + cells (C) were always kept at pH 7.4. AP enhanced CXCR4 mRNA levels (2.5-fold vs C; n=6; p&lt;0.05). In agreement with this result AP increased SDF-1-driven chemotaxis (2-fold vs C; n=6; p&lt;0.02) and transendothelial migration (10-fold vs C; n=5; p&lt;0.05) as well as SDF-1-driven differentiation toward the endothelial lineage as indicated by DiI-Ac-LDL uptake (2.2-fold vs C; n=10; p&lt;0.005), vWF positivity (2.2-fold vs C; n=4; p&lt;0.005) and KDR positivity (3-fold vs C; n=4; p&lt;0.005). The effect of AP on ckit + cell therapeutic potential was examined in a mouse model of hindlimb ischemia. Either AP or C cells (5 × 10 5 ) were injected into the adductor muscle at the time of femoral artery dissection. AP cells accelerated blood flow recovery vs C cells as determined by laser doppler perfusion imaging at different time points throughout the 14-day time course following acute ischemia (n=8; p&lt;0.01 vs C). Further, 14 days after surgery, AP cells increased capillary (1.3-fold vs C; n=6; p&lt;0.01) and arteriole number (4.6-fold vs C; n=6; p&lt;0.01) as well as the number of regenerating muscle fibers measured 7 days after surgery (4.4-fold vs C; n=6; p&lt;0.05). Conclusions. ckit + cells exposed to AP exhibit enhanced CXCR4 expression and SDF-1-directed cell responses in vitro and regenerative properties in a mouse model of hindlimb ischemia. Thus, AP may represent a simple, unexpensive and clinically applicable strategy to improve the therapeutic efficacy of bone marrow-derived cell transplantation.

Targeted delivery of cisplatin to prostate cancer cells by aptamer functionalized Pt(IV) prodrug-PLGA–PEG nanoparticles

Cisplatin is used to treat a variety of tumors, but dose limiting toxicities or intrinsic and acquired resistance limit its application in many types of cancer including prostate. We report a unique strategy to deliver cisplatin to prostate cancer cells by constructing Pt(IV)-encapsulated prostate-specific membrane antigen (PSMA) targeted nanoparticles (NPs) of poly(D,L-lactic-co-glycolic acid) (PLGA)-poly(ethylene glycol) (PEG)-functionalized controlled release polymers. By using PLGA-b-PEG nanoparticles with PSMA targeting aptamers (Apt) on the surface as a vehicle for the platinum(IV) compound c,t,c-[Pt(NH(3))(2)(O(2)CCH(2)CH(2)CH(2)CH(2)CH(3))(2)Cl(2)] (1), a lethal dose of cisplatin was delivered specifically to prostate cancer cells. PSMA aptamer targeted delivery of Pt(IV) cargos to PSMA(+) LNCaP prostate cancer cells by endocytosis of the nanoparticle vehicles was demonstrated using fluorescence microscopy by colocalization of green fluorescent labeled cholesterol-encapsulated NPs and early endosome marker EEA-1. The choice of linear hexyl chains in 1 was the result of a systematic study to optimize encapsulation and controlled release from the polymer without compromising either feature. Release of cisplatin from the polymeric nanoparticles after reduction of 1 and formation of cisplatin 1,2-intrastrand d(GpG) cross-links on nuclear DNA was confirmed by using a monoclonal antibody for the adduct. A comparison between the cytotoxic activities of Pt(IV)-encapsulated PLGA-b-PEG NPs with the PSMA aptamer on the surface (Pt-NP-Apt), cisplatin, and the nontargeted Pt(IV)-encapsulated NPs (Pt-NP) against human prostate PSMA-overexpressing LNCaP and PSMA(-) PC3 cancer cells revealed significant differences. The effectiveness of PSMA targeted Pt-NP-Apt nanoparticles against the PSMA(+) LNCaP cells is approximately an order of magnitude greater than that of free cisplatin.

COMMITTED ADIPOGENIC PROGENITORS ARE RESPONSIBLE FOR NOVEL ADIPOCYTE GENERATION IN RESPONSE TO DIETARY STIMULI

During adult life white fat depots can expand via hyperplasia and adipocyte infiltration can appear in aging or disease of non-adipose tissues. These observations suggest that stem/progenitor cells exist that can respond to a variety of stimuli to generate new adipocytes. We used a combination of surface markers to prospectively identify a population of cells containing all adipogenic colony-forming potential in skeletal muscle, subcutaneous and perigonadal adipose depots (adipogenic progenitors, APs). We did not observe any generation of cartilage, bone or muscle from AP cultures, suggesting that APs are committed to the adipocyte lineage. Confocal- and immunoelectron- microscopy analysis on tissues from BrdU-treated high fat diet-fed animals confirms the production of new adipocytes during weight gain. APs appear to participate in this response in a depot- specific manner. Furthermore, we observed a significant correlation between the size of the fat pad and frequency of actively cycling AP cells. Taken together, we show that we have identified an abundant population of lineage-restricted adipogenic progenitors. Importantly, our data suggests that during weight gain, new adipose generation relies on proliferation of these cells. AJ is supported by a Vancouver Coastal Health–CIHR–UBC MD/PhD Studentship Award.

Interrelation between different death mechanisms in anoxia–reoxygenation of neonatal rat cardiomyocytes

It is usually believed that apoptosis (Ap) and autophagy (Au) increases cell death during myocardial ischemia–reperfusion. In these experiments we have tested this assumption. Primary cultures of neonatal rat cardiomyocytes in our experiments contain 90.0±2.8% living (L), 3.3±1.09 necrotic (N), and 5.5±2.27 apoptotic (Ap) cells. After 30 min of anoxia (A) and 60 min reoxygenation (R) we have: L — 78.5±5.31%, N — 7.8±3.06% and Ap — 12.8±5.13 (P<0.05). After inhibition of apoptotic pathways by caspase-3 inhibitor N-acetyl-Asp-Glu-Val-Asp-al (DEVD, 100 μM) we observed that live cell population was not increased, but contrarily was decreased after A-R.