Transplantation In Immunodeficient Mice Research Articles

Haploinsufficiency at the CX3CR1 locus of hematopoietic stem cells favors the appearance of microglia-like cells in the central nervous system of transplant recipients

Transplantation of engineered hematopoietic stem/progenitor cells (HSPCs) showed curative potential in patients affected by neurometabolic diseases treated in early stage. Favoring the engraftment and maturation of the engineered HSPCs in the central nervous system (CNS) could allow enhancing further the therapeutic potential of this approach. Here we unveil that HSPCs haplo-insufficient at the Cx3cr1 (Cx3cr1−/+) locus are favored in central nervous system (CNS) engraftment and generation of microglia-like progeny cells (MLCs) as compared to wild type (Cx3cr1+/+) HSPCs upon transplantation in mice. Based on this evidence, we have developed a CRISPR-based targeted gene addition strategy at the human CX3CR1 locus resulting in an enhanced ability of the edited human HSPCs to generate mature MLCs upon transplantation in immunodeficient mice, and in lineage specific, regulated and robust transgene expression. This approach, which benefits from the modulation of pathways involved in microglia maturation and migration in haplo-insufficient cells, may broaden the application of HSPC gene therapy to a larger spectrum of neurometabolic and neurodegenerative diseases.

Loss of TET2 in human hematopoietic stem cells alters the development and function of neutrophils

Somatic mutations commonly occur in hematopoietic stem cells (HSCs). Some mutant clones outgrow through clonal hematopoiesis (CH) and produce mutated immune progenies shaping host immunity. Individuals with CH are asymptomatic but have an increased risk of developing leukemia, cardiovascular and pulmonary inflammatory diseases, and severe infections. Using genetic engineering of human HSCs (hHSCs) and transplantation in immunodeficient mice, we describe how a commonly mutated gene in CH, TET2, affects human neutrophil development and function. TET2 loss in hHSCs produce a distinct neutrophil heterogeneity in bone marrow and peripheral tissues by increasing the repopulating capacity of neutrophil progenitors and giving rise to low-granule neutrophils. Human neutrophils that inherited TET2 mutations mount exacerbated inflammatory responses and have more condensed chromatin, which correlates with compact neutrophil extracellular trap (NET) production. We expose here physiological abnormalities that may inform future strategies to detect TET2-CH and prevent NET-mediated pathologies associated with CH.

Large-scale engineering of hiPSC-derived nephron sheets and cryopreservation of their progenitors

BackgroundThe generation of human induced pluripotent stem cells (hiPSCs) has opened a world of opportunities for stem cell-based therapies in regenerative medicine. Currently, several human kidney organoid protocols are available that generate organoids containing kidney structures. However, these kidney organoids are relatively small ranging up to 0.13 cm2 and therefore contain a small number of nephrons compared to an adult kidney, thus defying the exploration of future use for therapy.MethodWe have developed a scalable, easily accessible, and reproducible protocol to increase the size of the organoid up to a nephron sheet of 2.5 cm2 up to a maximum of 12.6 cm2 containing a magnitude of nephrons.ResultsConfocal microscopy showed that the subunits of the nephrons remain evenly distributed throughout the entire sheet and that these tissue sheets can attain ~ 30,000–40,000 glomerular structures. Upon transplantation in immunodeficient mice, such nephron sheets became vascularized and matured. They also show reuptake of injected low-molecular mass dextran molecules in the tubular structures, indicative of glomerular filtration. Furthermore, we developed a protocol for the cryopreservation of intermediate mesoderm cells during the differentiation and demonstrate that these cells can be successfully thawed and recovered to create such tissue sheets.ConclusionThe scalability of the procedures, and the ability to cryopreserve the cells during differentiation are important steps forward in the translation of these differentiation protocols to future clinical applications such as transplantable auxiliary kidney tissue.

A negative feedback loop between fibroadipogenic progenitors and muscle fibres involving endothelin promotes human muscle fibrosis.

BackgroundFibrosis is defined as an excessive accumulation of extracellular matrix (ECM) components. Many organs are subjected to fibrosis including the lung, liver, heart, skin, kidney, and muscle. Muscle fibrosis occurs in response to trauma, aging, or dystrophies and impairs muscle function. Fibrosis represents a hurdle for the treatment of human muscular dystrophies. While data on the mechanisms of fibrosis have mostly been investigated in mice, dystrophic mouse models often do not recapitulate fibrosis as observed in human patients. Consequently, the cellular and molecular mechanisms that lead to fibrosis in human muscle still need to be identified.MethodsCombining mass cytometry, transcriptome profiling, in vitro co‐culture experiments, and in vivo transplantation in immunodeficient mice, we investigated the role and nature of nonmyogenic cells (fibroadipogenic progenitors, FAPs) from human fibrotic muscles of healthy individuals (FibMCT) and individuals with oculopharyngeal muscular dystrophy (OPMD; FibMOP), as compared with nonmyogenic cells from human nonfibrotic muscle (MCT).ResultsWe found that the proliferation rate of FAPs from fibrotic muscle is 3–4 times higher than those of FAPs from nonfibrotic muscle (population doubling per day: MCT 0.2 ± 0.1, FibMCT 0.7 ± 0.1, and FibMOP 0.8 ± 0.3). When cocultured with muscle cells, FAPs from fibrotic muscle impair the fusion index unlike MCT FAPs (myoblasts alone 57.3 ± 11.1%, coculture with MCT 43.1 ± 8.9%, with FibMCT 31.7 ± 8.2%, and with FibMOP 36.06 ± 10.29%). We also observed an increased proliferation of FAPs from fibrotic muscles in these co‐cultures in differentiation conditions (FibMCT +17.4%, P < 0.01 and FibMOP +15.1%, P < 0.01). This effect is likely linked to the increased activation of the canonical TGFβ‐SMAD pathway in FAPs from fibrotic muscles evidenced by pSMAD3 immunostaining (P < 0.05). In addition to the profibrogenic TGFβ pathway, we identified endothelin as a new actor implicated in the altered cross‐talk between muscle cells and fibrotic FAPs, confirmed by an improvement of the fusion index in the presence of bosentan, an endothelin receptor antagonist (from 33.8 ± 10.9% to 52.9 ± 10.1%, P < 0.05).ConclusionsOur data demonstrate the key role of FAPs and their cross‐talk with muscle cells through a paracrine signalling pathway in fibrosis of human skeletal muscle and identify endothelin as a new druggable target to counteract human muscle fibrosis.

In vitro elastic cartilage reconstruction using human auricular perichondrial chondroprogenitor cell–derived micro 3D spheroids

Morphologically stable scaffold-free elastic cartilage tissue is crucial for treating external ear abnormalities. However, establishing adequate mechanical strength is challenging, owing to the difficulty of achieving chondrogenic differentiation in vitro; thus, cartilage reconstruction is a complex task. Auricular perichondrial chondroprogenitor cells exhibit high proliferation potential and can be obtained with minimal invasion. Therefore, these cells are an ideal resource for elastic cartilage reconstruction. In this study, we aimed to develop a novel in vitro scaffold-free method for elastic cartilage reconstruction, using human auricular perichondrial chondroprogenitor cells. Inducing chondrogenesis by using microscopic spheroids similar to auricular hillocks significantly increased the chondrogenic potential. The size and elasticity of the tissue were maintained after craniofacial transplantation in immunodeficient mice, suggesting that the reconstructed tissue was morphologically stable. Our novel tissue reconstruction method may facilitate the development of future treatments for external ear abnormalities.

Abstract 501: Aldh2 mutation promotes oral and esophageal cancer stem cells via autophagy-mediated redox homeostasis

Abstract Introduction: Heavy alcohol (EtOH) drinking and tobacco smoking increase the risk for upper digestive (e.g. oral and esophageal) squamous cell carcinomas (SCC) in individuals with polymorphic mutations in aldehyde dehydrogenase 2 (Aldh2). Mutant Aldh2E487K protein delays mitochondrial clearance of acetaldehyde, the chief metabolite of EtOH and constituents of tobacco smoke, aka a major human carcinogen. The role of Aldh2 in tumor biology remains elusive. Methods: Single cell-derived three-dimensional (3D) organoids were generated from murine oral and esophageal preneoplastic and SCC lesions carrying wild-type Aldh2WT or mutant Aldh2E487K as well as human SCC cell lines with Aldh2E487K, the latter subjected to xenograft transplantation in immunodeficient mice. 3D organoids and xenograft tumors were analyzed by morphology and flow cytometry to determine CD44, autophagy (cyto-ID), proliferation (EdU), and reactive oxygen species (ROS) following EtOH exposure ex vivo (0.1-2% EtOH) or in vivo (10% EtOH in drinking water) along with or without inhibitors of alcohol metabolism (4-methylpyrazole; 4MP, siRNA against Adh1B and Cyp2E1), and autophagy flux (chloroquine; CQ or siRNA against Atg7). Flow cytometry was performed to determine cancer stem-like cells with high CD44 expression (CD44H cells). Results: EtOH stimulated xenograft tumor growth and increased highly proliferative intratumoral CD44H cells with increased autophagy. EtOH-stimulated tumor growth and induction of CD44H cells were sharply suppressed by concurrent 4MP treatment, indicating that EtOH is directly metabolized in SCC cells to promote tumor growth via CD44H cells. These EtOH-induced effects were recapitulated in 3D organoids where Aldh2E487K augmented EtOH-mediated induction of CD44H cells and organoid formation with increased cellular atypia. EtOH induced mitochondrial superoxide and ROS-dependent autophagy activation in CD44H cells. Inhibition of autophagy prevented EtOH from enriching CD44H cells in 3D organoids, suggesting that autophagy may limit alcohol-induced oxidative stress as a cytoprotective mechanism. Conclusions: These studies provide not only mechanistic insights into the role of Aldh2 mutation in oral and esophageal SCC pathogenesis but also establish the utility of the 3D organoid system to test the functional interplay between genetic and environmental factors which may subsequently be targeted for pharmacological intervention in the setting of personalized medicine. Citation Format: Masataka Shimonosono, Takashi Kijima, Hisatsugu Maekawa, Satoshi Takada, Koji Tanaka, Varun Sahu, Manti Guha, Andres J. Klein-Szanto, J. Alan Diehl, Adam J. Bass, Anil K. Rustgi, Elizabeth Philipone, Angela J. Yoon, Hiroshi Nakagawa. Aldh2 mutation promotes oral and esophageal cancer stem cells via autophagy-mediated redox homeostasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 501.

Targeting Salt-Inducible Kinase 3 As a Therapeutic Approach for Acute Myeloid Leukemia

Acute myeloid leukemia (AML) remains a devastating illness, with a clear need for the development of novel anti-leukemic therapy. CRISPR-Cas9 functional genomic screens have revealed hundreds of genetic vulnerabilities in AML cells, which can be leveraged to nominate clinically actionable targets. Our previous CRISPR screens in AML cell lines identified Salt-Inducible Kinase 3 (SIK3) as an essential gene for a subset of AML cells with high expression of the oncogenic transcription factor MEF2C, such as AMLs harboring Mixed Lineage Leukemia (MLL)-fusion proteins. Mechanistically, we have shown that the maintenance of MEF2C transcriptional output requires SIK3, which phosphorylates histone deacetylase 4 (HDAC4), a repressive cofactor of MEF2C, and thus prevents HDAC4 from nuclear entry and from suppressing MEF2C activity. SIKs 1-3 are serine/threonine kinases belonging to the adenosine monophosphate-activated protein kinase (AMPK) family. The physiological function of SIKs has been investigated in the regulation of gluconeogenesis, myogenesis and osteogenesis, yet the critical role of SIK3 in AML growth is just emerging. Thus, the evaluation of its translational potential in relevant pre-clinic models, such as patient-derived-xenografts (PDXs), is necessary. To do so, we have established a novel MLL-AF9 PDX model tractable for CRISPR genome editing via lentivirally expressing Cas9 and serial transplantation in immunodeficient mice. The PDX model maintains stable Cas9 activity; knockout of SIK3 hampered leukemia growth in vivo, demonstrating the utility of this model and the functional significance of SIK3 in AML. In parallel, we tested the anti-leukemic effect of the tool compound YKL-05-099, which inhibits the SIK kinase family and has a suitable bioavailability in mice. YKL-05-099 treatment inhibited the growth of AML cell lines in vitro via inducing cell cycle arrest and apoptosis, and AML cells with high MEF2C protein expression tended to be more sensitive to YKL-05-099. YKL-05-099 was well-tolerated by mice, and after daily administration for a month, no obvious toxicities or physiological abnormalities were observed. Normal hematopoiesis was also sustained, as indicated by complete blood counts and lineage flow cytometry analysis. By contrast, a two-week treatment regimen successfully attenuated disease progression in vivo and extended animal survival in both an MLL-AF9 mouse AML and PDX models. Importantly, we have confirmed the action of the compound is via on-target inhibition of the SIK3-HDAC4-MEF2C axis. Western-blot analysis revealed that YKL-05-099 treatment led to rapid dephosphorylation and nuclear accumulation of HDAC4 in AML cells. Accordingly, similar to SIK3 knockout, ChIP-seq analysis showed that YKL-05-099 selectively reduced histone H3 lysine 27 acetylation at a small number of genomic locations, which are enriched for MEF2C occupancy sites. This finding was supported by RNA-seq analysis as well, which showed that YKL-05-099 treatment suppressed a similar transcriptional program as observed following genetic targeting of SIK3 or MEF2C. Additionally, either overexpressing a SIK3 gatekeeper mutant or deleting HDAC4 in MLL-fusion AML cells can attenuate their sensitivity to YKL-05-099. High MEF2C expression in AML is associated with poor response to conventional chemotherapy. Our findings validate SIK3 inhibition as a possible therapeutic approach in MEF2C-positive AML and provide a rationale for developing improved drug-like inhibitors of SIK3, with enhanced specificity and bioactivity, for definitive pre-clinical investigation and for future studies in patients with AML. Disclosures Lin: Leukemia &amp; Lymphoma Society: Research Funding. Tarumoto:Lauri Strauss Leukemia Foundation: Research Funding. Gray:Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Vakoc:Boehringer-Ingelheim: Research Funding; KSQ Therapeutics: Consultancy. Stegmaier:Rigel Pharmaceuticals: Consultancy; Novartis: Research Funding.

Cdh4 Down-Regulation Impairs in Vivo Infiltration and Malignancy in Patients Derived Glioblastoma Cells.

The high invasive phenotype of glioblastoma is one of the main causes of therapy inefficacy and tumor relapse. Cell adhesion molecules of the cadherin family are involved in cell migration and are known as master regulators of epithelial tumor invasiveness, but their role in glioblastoma is less understood. In particular, we recently demonstrated, in the syngeneic murine model, the occurrence of a previously undescribed cadherin switch between Cdh2 and Cdh4 during gliomagenesis, which is necessary for the acquisition of the highly infiltrative and tumorigenic phenotype of these cells. In the present study, we tested the role of Cdh4 in human gliomas. Our results on patient-derived glioma cells demonstrate a positive correlation between Cdh4 expression levels and the loss of cell–cell contact inhibition of proliferation controls that allows cells to proliferate over confluence. Moreover, the silencing of Cdh4 by artificial microRNAs induced a decrease in the infiltrative ability of human glioma cells both in vitro and in vivo. More strikingly, Cdh4 silencing induced an impairment of the tumorigenic potential of these cells after orthotopic transplantation in immunodeficient mice. Overall, we conclude that in human glioblastoma, Cdh4 can also actively contribute in regulating cell invasiveness and malignancy.

Abstract 161: TRIM21 suppresses invasion of breast cancer cells

Abstract Trim21 is an E3 ligase that is involved in the regulation of metastasis. High-level expression of TRIM21 is associated with good prognosis of breast cancer. After transplantation in immunodeficient mice, TRIM21-depleted MCF7 cells form tumors that show evident local invasion, i.e., extensive infiltration of single tumor cells into nearby muscle fibers. Knockdown of TRIM21 increases the migration and invasion of MCF7 and T47D cells by altering the expression of genes that regulate epithelial to mesenchymal transition(EMT). TRIM21 interacts with Snail, a master regulator of EMT, and the B-Box domain of TRIM21 is essential for the interaction between TRIM21 and Snail. Overexpression of TRIM21 leads to increased ubiquitination and subsequent proteosomal degradation of Snail, while depletion of TRIM21 decreases the ubiquitination and degradation of Snail. Most Importantly, knockdown of Snail suppresses the increased migration and invasion of MCF7 and T47D cells caused by TRIM21 knockdown. Collectively, our study demonstrates that TRIM21 modulates EMT by mediating the stability of Snail in breast cancer cells. Citation Format: Xin Hu, Yue Jin, Yu Zhang, Boyuan Li, Youzhong Wan. TRIM21 suppresses invasion of breast cancer cells [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 161.

Differentiation of Human Embryonic Stem Cells into Engrafting Myogenic Precursor Cells

Degenerative muscle diseases affect muscle tissue integrity and function. Human embryonic stem cells (hESC) are an attractive source of cells to use in regenerative therapies due to their unlimited capacity to divide and ability to specialize into a wide variety of cell types. A practical way to derive therapeutic myogenic stem cells from hESC is lacking. In this study, we demonstrate the development of two serum-free conditions to direct the differentiation of hESC towards a myogenic precursor state. Using TGFß and PI3Kinase inhibitors in combination with bFGF we showed that one week of differentiation is sufficient for hESC to specialize into PAX3+/PAX7+ myogenic precursor cells. These cells also possess the capacity to further differentiate in vitro into more specialized myogenic cells that express MYOD, Myogenin, Desmin and MYHC, and showed engraftment in vivo upon transplantation in immunodeficient mice. Ex vivo myomechanical studies of dystrophic mouse hindlimb muscle showed functional improvement one month post-transplantation. In summary, this study describes a promising system to derive engrafting muscle precursor cells solely using chemical substances in serum-free conditions and without genetic manipulation.

Cardiac Progenitors Induced from Human Induced Pluripotent Stem Cells with Cardiogenic Small Molecule Effectively Regenerate Infarcted Hearts and Attenuate Fibrosis.

Cardiac progenitor cells (CPCs) being multipotent offer a promising source for cardiac repair due to their ability to proliferate and multiply into cardiac lineage cells. Here, we explored a novel strategy for human CPCs generation from human induced pluripotent stem cells (hiPSCs) using a cardiogenic small molecule, isoxazole (ISX-9) and their ability to grow in the scar tissue for functional improvement in the infarcted myocardium. CPCs were induced from hiPSCs with ISX-9. CPCs were characterized by immunocytochemistry and RT-PCR. The CPC survival and differentiation in the infarcted hearts were determined by in vivo transplantation in immunodeficient mice following left anterior descending artery ligation and their effects were determined on fibrosis and functional improvement. ISX-9 simultaneously induced expression of cardiac transcription factors, NK2 homeobox 5, islet-1, GATA binding protein 4, myocyte enhancer factor-2 in hiPSCs within 3 days of treatment and successfully differentiated into three cardiac lineages in vitro. Messenger RNA and microRNA-sequencing results showed that ISX-9 targeted multiple cardiac differentiation, proliferation signaling pathways and upregulated myogenesis and cardiac hypertrophy related-microRNA. ISX-9 activated multiple pathways including transforming growth factor β induced epithelial-mesenchymal transition signaling, canonical, and non-canonical Wnt signaling at different stages of cardiac differentiation. CPCs transplantation promoted myoangiogenesis, attenuated fibrosis, and led to functional improvement in treated mice.

Functional Maturation and In Vitro Differentiation of Neonatal Porcine Islet Grafts

There is a strong rationale to pursue the use of neonatal porcine islets (NPIs) as an unlimited source of islets for clinical xenotransplantation. Because NPIs are composed of immature insulin producing beta (ß) cells and ductal precursor cells, they provide an ideal model to examine culture conditions to enhance ß cell proliferation and/or ß cell neoformation from ductal cells. In an attempt to optimize the potential of NPIs as a source of ß cell grafts, we used an in vitro differentiation protocol and measured its effect on the functional maturation and differentiation of NPIs. Pancreata from 1- to 3-day-old neonatal pigs were digested and cultured in standard Ham's F10 media for 5 days. Each independent preparation was then further cultured in Dulbecco's modified Eagle medium nutrient mixture-F12 differentiation media containing growth factors added in a stepwise fashion, or cultured in control Ham's F10 media. After 20 days in culture, islets were assessed for insulin secretory capacity, cellular composition, gene expression, and metabolic activity after transplantation in immunodeficient mice with diabetes. Compared with control islets, differentiated islets exhibited a significantly higher proportion of endocrine cells, proliferating cell nuclear antigen double positive ß cells, and an enhanced glucose-stimulated insulin secretory activity. Mice transplanted with differentiated islets had significantly lower blood glucose values at weeks 18 and 20 compared with nondifferentiated controls and were shown to be more glucose tolerant. Culturing NPIs in a 20-day stepwise differentiation media increases the proportion of endocrine cells and augments both in vitro and in vivo function of the islets.

Abstract LB-363: Characterization of in vitro and in vivo models generated from disseminated tumor cells of patients without manifest metastasis

Abstract We recently showed that dissemination of few melanoma cells to the lymph nodes is a quantitative risk factor for death from melanoma. However, the analysis of disseminated tumor cells (DTC) is limited by their extremely low frequency, resulting in a lack of in vitro/in vivo models for mechanistic studies of early systemic disease. Therefore, the objective of this study was to develop protocols to expand single DTC from lymph nodes of melanoma patients to establish better preclinical models for adjuvant cancer therapies. Sentinel lymph nodes from melanoma patients were cut in halves, disaggregated and analyzed for the presence of gp100-positive DTC. The other half was used for diagnostic assessment by histopathology. Single DTC within the lymph node-derived single cell suspension were then propagated under specific sphere forming conditions and expanded by transplantation in immunodeficient mice. DTC based pre-clinical in vitro/in vivo models were then characterized on molecular level and were used for functional analyses. To test drugs on DTC in presence of a human immune system we additionally generated humanized xenograft models. We successfully established cell lines and xenografts from DTCs of 17 melanoma patients. We confirmed the origin of the cells by genomic fingerprint and analyzed the genomic profile of the expanded cells over various culture and animal passages. DTC cell lines were used for functional analysis and for testing the efficacy of targeted therapies. In addition, we set up a novel preclinical mouse model based on expanded melanoma DTC. For this, we established a human immune system in immunodeficient mice, which were then transplanted with melanoma DTC-derived cells. Interestingly, the presence of a human immune system significantly induced tumor formation, as well as dissemination of melanoma cells and improved the response to a targeted therapy compared to mice without a humanized immune system. The development of DTC based pre-clinical in vitro/in vivo models enables mechanistic studies on DTCs and drug tests against the target cells of adjuvant therapies in the presence of human immune cells. These models may help to identify candidate adjuvant therapies targeting DTCs and to understand mechanisms of drug resistance of currently applied targeted therapies. Citation Format: Christian Werno, Kathrin Weidele, Steffi Treitschke, Catherine Botteron, Sebastian Scheitler, Bernhard Polzer, Melanie Werner-Klein, Christoph Andreas Klein. Characterization of in vitro and in vivo models generated from disseminated tumor cells of patients without manifest metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-363.

PO-440 Development and characterisation of pre-clinical in vitro/in vivo models from disseminated tumour cells of melanoma patients

IntroductionWe recently showed that dissemination of few melanoma cells to the lymph nodes is a quantitative risk factor for death from melanoma. However, the analysis of disseminated tumour cells (DTC) is limited by their extremely low frequency, resulting in a lack of in vitro/in vivo models for mechanistic studies of early systemic disease. Therefore, the objective of this study was to develop protocols to expand single DTC from lymph nodes of melanoma patients to establish better preclinical models for adjuvant cancer therapies.Material and methodsSentinel lymph nodes from melanoma patients were cut in halves, disaggregated and analysed for the presence of gp100-positive DTC. The other half was used for diagnostic assessment by histopathology. Single DTC within the lymph node-derived single cell suspension were then propagated under specific sphere forming conditions and expanded by transplantation in immunodeficient mice. DTC based pre-clinical in vitro/in vivo models were then characterised on molecular level and were used for functional analyses. To test drugs on DTC in presence of a human immune system we additionally generated humanised xenograft models.Results and discussionsWe successfully established cell lines and xenografts from DTCs of 17 melanoma patients. We confirmed the origin of the cells by genomic fingerprint and analysed the genomic profile of the expanded cells over various culture and animal passages. DTC cell lines were used for functional analysis and for testing the efficacy of targeted therapies. In addition, we set up a novel preclinical mouse model based on expanded melanoma DTC. For this, we established a human immune system in immunodeficient mice, which were then transplanted with melanoma DTC-derived cells. Interestingly, the presence of a human immune system significantly induced tumour formation, as well as dissemination of melanoma cells and improved the response to a targeted therapy compared to mice without a humanised immune system.ConclusionThe development of DTC based pre-clinical in vitro/in vivo models enables mechanistic studies on DTCs and drug tests against the target cells of adjuvant therapies in the presence of human immune cells. These models may help to identify candidate adjuvant therapies targeting DTCs and to understand mechanisms of drug resistance of currently applied targeted therapies.

β-Cell Replacement in Mice Using Human Type 1 Diabetes Nuclear Transfer Embryonic Stem Cells.

β-Cells derived from stem cells hold great promise for cell replacement therapy for diabetes. Here we examine the ability of nuclear transfer embryonic stem cells (NT-ESs) derived from a patient with type 1 diabetes to differentiate into β-cells and provide a source of autologous islets for cell replacement. NT-ESs differentiate in vitro with an average efficiency of 55% into C-peptide-positive cells, expressing markers of mature β-cells, including MAFA and NKX6.1. Upon transplantation in immunodeficient mice, grafted cells form vascularized islet-like structures containing MAFA/C-peptide-positive cells. These β-cells adapt insulin secretion to ambient metabolite status and show normal insulin processing. Importantly, NT-ES-β-cells maintain normal blood glucose levels after ablation of the mouse endogenous β-cells. Cystic structures, but no teratomas, were observed in NT-ES-β-cell grafts. Isogenic induced pluripotent stem cell lines showed greater variability in β-cell differentiation. Even though different methods of somatic cell reprogramming result in stem cell lines that are molecularly indistinguishable, full differentiation competence is more common in ES cell lines than in induced pluripotent stem cell lines. These results demonstrate the suitability of NT-ES-β-cells for cell replacement for type 1 diabetes and provide proof of principle for therapeutic cloning combined with cell therapy.

Abstract 2239: Establishment of lung cancer CTCs competent for lung metastasis

Abstract Circulating tumor cells (CTCs) have been implicated as a population of cells that may seed metastasis, which led to the 90% of dead in cancer patients. Though the detection technologies has been developing swiftly, how to culture the cells successfully and established a permanent cell line are the bottleneck in the research of metastasis mechanism. Here, we report, for the first time, the establishment of permanent cell line from CTCs of one Non-small cell lung cancer patient and identifying the biological characterization. The cell line designated CTC-0430 has been cultured for more than one and half years, and the cells have been characterized at the genome, proliferation, drug resistance, the expression of surface marker, cytokine secretion level and the characterization of tumor formation in vivo by immunohistochemisty and immunofluorescence. This thorough analysis showed that CTC-0430 cells display a stable phenotype characterized by an intermediate epithelial/mesenchymal phenotype, stem-cell like properties and the drug sensitivity test indicating stronger drug resistance compared to A549 and 95-D. When compare with A549 and 95-D cells, CTC-0430 cells high expression of ICAM-1 and ICAM-3, low expression of CX3CL1 and CXCL1. Functional studies showed that CTC-0430 cells induced rapidly in vivo tumors after subcutaneous transplantation in immunodeficient mice, and the lung metastasis were found after six months later. Besides, three months after we injected the cells into the caudal vein of NOD/SCID mice, the lung metastasis were formed. And then the metastasis lung tissue were used to make IHC and IF, which indicated that the biological characterizations of this cell line are different from A549. The establishment stable cell line of lung cancer CTCs can provide in vivo research platform for the intervention study of postoperative metastasis of lung cancer. Citation Format: Zujun Que, Bin Luo, Qihui Shi, Jianhui Tian. Establishment of lung cancer CTCs competent for lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2239. doi:10.1158/1538-7445.AM2017-2239

Human myogenic reserve cells are quiescent stem cells that contribute to muscle regeneration after intramuscular transplantation in immunodeficient mice

Satellite cells, localized within muscles in vivo, are Pax7+ muscle stem cells supporting skeletal muscle growth and regeneration. Unfortunately, their amplification in vitro, required for their therapeutic use, is associated with reduced regenerative potential. In the present study, we investigated if human myogenic reserve cells (MRC) obtained in vitro, represented a reliable cell source for muscle repair. For this purpose, primary human myoblasts were freshly isolated and expanded. After 2 days of differentiation, 62 ± 2.9% of the nuclei were localized in myotubes and 38 ± 2.9% in the mononucleated non-fusing MRC. Eighty percent of freshly isolated human MRC expressed a phenotype similar to human quiescent satellite cells (CD56+/Pax7+/MyoD−/Ki67− cells). Fourteen days and 21 days after cell transplantation in immunodeficient mice, live human cells were significantly more numerous and the percentage of Pax7+/human lamin A/C+ cells was 2 fold higher in muscles of animals injected with MRC compared to those injected with human myoblasts, despite that percentage of spectrin+ and lamin A/C+ human fibers in both groups MRC were similar. Taken together, these data provide evidence that MRC generated in vitro represent a promising source of cells for improving regeneration of injured skeletal muscles.

GMP-conformant on-site manufacturing of a CD133+ stem cell product for cardiovascular regeneration

BackgroundCD133+ stem cells represent a promising subpopulation for innovative cell-based therapies in cardiovascular regeneration. Several clinical trials have shown remarkable beneficial effects following their intramyocardial transplantation. Yet, the purification of CD133+ stem cells is typically performed in centralized clean room facilities using semi-automatic manufacturing processes based on magnetic cell sorting (MACS®). However, this requires time-consuming and cost-intensive logistics.MethodsCD133+ stem cells were purified from patient-derived sternal bone marrow using the recently developed automatic CliniMACS Prodigy® BM-133 System (Prodigy). The entire manufacturing process, as well as the subsequent quality control of the final cell product (CP), were realized on-site and in compliance with EU guidelines for Good Manufacturing Practice. The biological activity of automatically isolated CD133+ cells was evaluated and compared to manually isolated CD133+ cells via functional assays as well as immunofluorescence microscopy. In addition, the regenerative potential of purified stem cells was assessed 3 weeks after transplantation in immunodeficient mice which had been subjected to experimental myocardial infarction.ResultsWe established for the first time an on-site manufacturing procedure for stem CPs intended for the treatment of ischemic heart diseases using an automatized system. On average, 0.88 × 106 viable CD133+ cells with a mean log10 depletion of 3.23 ± 0.19 of non-target cells were isolated. Furthermore, we demonstrated that these automatically isolated cells bear proliferation and differentiation capacities comparable to manually isolated cells in vitro. Moreover, the automatically generated CP shows equal cardiac regeneration potential in vivo.ConclusionsOur results indicate that the Prodigy is a powerful system for automatic manufacturing of a CD133+ CP within few hours. Compared to conventional manufacturing processes, future clinical application of this system offers multiple benefits including stable CP quality and on-site purification under reduced clean room requirements. This will allow saving of time, reduced logistics and diminished costs.

CD56+ Muscle Derived Cells but Not Retinal NG2+ Perivascular Cells of Nonhuman Primates are Myogenic after Intramuscular Transplantation in Immunodeficient Mice

Some reports attributed to pericytes and other perivascular cells (PCs), regardless of their origin, optimal properties for cell therapy in myology. The retina is an ideal tissue to obtain pericytes and one study reported that PCs from the mouse retina were myogenic in vitro. Given the importance of nonhuman primates (NHPs) for translational research, we compared the in vivo myogenicity of NHP retinal PCs and satellite cell derived myoblasts (SCDMs) by transplantation in immunodeficient mice. We used a protocol to culture retinal pericytes of large mammals with macaque retinas. By flow cytometry, 76%-78% of the cultured cells were NG2+. CD56+ SCDMs from another macaque were proliferated in vitro. Both Tibialis anterior muscles of 4 SCID mice were injected with 1x106 cells in saline (SCDMs in the right muscles and PCs in the left), using cardiotoxin to induce muscle regeneration. They were sampled 1 month later and analyzed by histology. In SCDM-grafted muscles, NHP nuclei were abundant, in large regions with numerous NHP-derived myofibers, and some of them were Pax7+. PC-grafted muscles showed no muscle regeneration, have few NHP nuclei in small regions devoid of myofibers, and no NHP-myofibers or Pax7+ NHP nuclei were observed. Therefore, NHP SCDMs, but not retinal NG2+ PCs, regenerated muscle in vivo in immunodeficient mice.

Heterogeneous leukemia stem cells in myeloid blast phase chronic myeloid leukemia

AbstractChronic myeloid leukemia (CML) is an excellent model of the multistep processes in cancer. Initiating BCR-ABL mutations are required for the initial phase of the disease (chronic phase, CP-CML). Some CP-CML patients acquire additional mutation(s) that transforms CP-CML to poor prognosis, hard to treat, acute myeloid or lymphoid leukemia or blast phase CML (BP-CML). It is unclear where in the hemopoietic hierarchy additional mutations are acquired in BP-CML, how the hemopoietic hierarchy is altered as a consequence, and the cellular identity of the resulting leukemia-propagating stem cell (LSC) populations. Here, we show that myeloid BP-CML is associated with expanded populations that have the immunophenotype of normal progenitor populations that vary between patients. Serial transplantation in immunodeficient mice demonstrated functional LSCs reside in multiple populations with the immunophenotype of normal progenitor as well as stem cells. Multicolor fluorescence in situ hybridization detected serial acquisition of cytogenetic abnormalities of chromosome 17, associated with transformation to BP-CML, that is detected with equal frequency in all functional LSC compartments. New effective myeloid BP-CML therapies will likely have to target all these LSC populations.