Establishment of patient-derived renal cell carcinoma (RCC) models based on orthotopic xenografts (PDX) and cancer stem cell (CSC) isolation to provide prognostic and predictive information.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e16055-e16055
Author(s):  
Simona di Martino ◽  
Gabriele de Luca ◽  
Ludovica Grassi ◽  
Giulia Federici ◽  
Laura De Salvo ◽  
...  
Keyword(s):  
Stem Cell ◽  
Drug Testing ◽  
Stage Iv ◽  
Predictive Biomarkers ◽  
Cell Populations ◽  
Type I ◽  
Prognostic Information ◽  
Lineage Differentiation ◽  
Immunocompromised Mice

e16055 Background: The introduction of VEGF/VEGFR- and mTOR, and immune checkpoint-targeted agents has radically changed metastatic RCC treatment; however, predictive biomarkers are still lacking and the potential for curative impact of such treatments in earlier disease stages is hotly debated. To identify potential diagnostic, prognostic, and predictive biomarkers, we developed patient-derived preclinical RCC models based on PDX obtained from freshly dissociated cancer tissues and combining isolation of cell populations endowed with self renewal and multi-lineage differentiation abilities (CSC). Methods: 182 surgical RCC specimens (clear cell, n = 110; papillary type I/II, n = 23, chromophobe/oncocytoma, n = 26, other, n = 23; stage I-III, n = 124, stage IV, n = 28, n/a n = 30) were collected; tumor spheroids were obtained and characterized in 57 cases, using a stem-cell isolating medium supplemented with EGF/b-FGF; 30 cancer samples were orthotopically injected in immunocompromised mice and were able to engraft in 67% of cases (G3-G4 cases more frequently than G1-G2 cases, p = 0.04). Results: PDX tumors recapitulated histological appearance, sarcomatoid features when they were present, and tumor heterogeneity of their human counterpart; interestingly, PDX engraftment was significantly correlated with shorter DFS in the corresponding patient population (p = 0.12). We conducted proteomic analysis by Reverse Phase Protein Array (RPPA) in 21 patient-derived CSC models in vitro: a panel of established (HIF, VEGFR, mTOR) and novel (EGFR(Y1148)), AKT, PI3K) oncogenic signals were deregulated in our isolated cell populations; molecular endpoints were correlated with grading and with angiogenesis and mTOR pathways (p < 0.05). Data analysis is ongoing to ascertain whether specific RPPA-signatures can potentially complement clinical prognostic information and suggest candidate biomarkers and potential targets for therapy. Conclusions: In the era of personalized therapy, the combined use of PDX and RPPA from tumor specimens may be very useful for drug testing and patient stratification in RCC.

scholarly journals Activation of two distinct Sox9-EGFP-expressing intestinal stem cell populations during crypt regeneration after irradiation

2012 ◽  
Vol 302 (10) ◽  
pp. G1111-G1132 ◽  
Author(s):  
Laurianne Van Landeghem ◽  
M. Agostina Santoro ◽  
Adrienne E. Krebs ◽  
Amanda T. Mah ◽  
Jeffrey J. Dehmer ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Populations ◽  
Intestinal Epithelial ◽  
P53 Signaling ◽  
Reporter Mice ◽  
Radiation Induced ◽  
Molecular Phenotypes ◽  
Stem Cell Populations ◽  
Induced Changes

Recent identification of intestinal epithelial stem cell (ISC) markers and development of ISC reporter mice permit visualization and isolation of regenerating ISCs after radiation to define their functional and molecular phenotypes. Previous studies in uninjured intestine of Sox9-EGFP reporter mice demonstrate that ISCs express low levels of Sox9-EGFP (Sox9-EGFP Low), whereas enteroendocrine cells (EEC) express high levels of Sox9-EGFP (Sox9-EGFP High). We hypothesized that Sox9-EGFP Low ISCs would expand after radiation, exhibit enhanced proliferative capacities, and adopt a distinct gene expression profile associated with rapid proliferation. Sox9-EGFP mice were given 14 Gy abdominal radiation and studied between days 3 and 9 postradiation. Radiation-induced changes in number, growth, and transcriptome of the different Sox9-EGFP cell populations were determined by histology, flow cytometry, in vitro culture assays, and microarray. Microarray confirmed that nonirradiated Sox9-EGFP Low cells are enriched for Lgr5 mRNA and mRNAs enriched in Lgr5-ISCs and identified additional putative ISC markers. Sox9-EGFP High cells were enriched for EEC markers, as well as Bmi1 and Hopx, which are putative markers of quiescent ISCs. Irradiation caused complete crypt loss, followed by expansion and hyperproliferation of Sox9-EGFP Low cells. From nonirradiated intestine, only Sox9-EGFP Low cells exhibited ISC characteristics of forming organoids in culture, whereas during regeneration both Sox9-EGFP Low and High cells formed organoids. Microarray demonstrated that regenerating Sox9-EGFP High cells exhibited transcriptomic changes linked to p53-signaling and ISC-like functions including DNA repair and reduced oxidative metabolism. These findings support a model in which Sox9-EGFP Low cells represent active ISCs, Sox9-EGFP High cells contain radiation-activatable cells with ISC characteristics, and both participate in crypt regeneration.

scholarly journals Improving the immunosuppressive potential of articular chondroprogenitors in a three-dimensional culture setting

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Guillermo Bauza ◽  
Anna Pasto ◽  
Patrick Mcculloch ◽  
David Lintner ◽  
Ava Brozovich ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Articular Cartilage ◽  
Cartilage Repair ◽  
Adipogenic Differentiation ◽  
3D Culture ◽  
Cell Populations ◽  
Stem Cell Markers ◽  
3D Scaffold

Abstract Cartilage repair in osteoarthritic patients remains a challenge. Identifying resident or donor stem/progenitor cell populations is crucial for augmenting the low intrinsic repair potential of hyaline cartilage. Furthermore, mediating the interaction between these cells and the local immunogenic environment is thought to be critical for long term repair and regeneration. In this study we propose articular cartilage progenitor/stem cells (CPSC) as a valid alternative to bone marrow-derived mesenchymal stem cells (BMMSC) for cartilage repair strategies after trauma. Similar to BMMSC, CPSC isolated from osteoarthritic patients express stem cell markers and have chondrogenic, osteogenic, and adipogenic differentiation ability. In an in vitro 2D setting, CPSC show higher expression of SPP1 and LEP, markers of osteogenic and adipogenic differentiation, respectively. CPSC also display a higher commitment toward chondrogenesis as demonstrated by a higher expression of ACAN. BMMSC and CPSC were cultured in vitro using a previously established collagen-chondroitin sulfate 3D scaffold. The scaffold mimics the cartilage niche, allowing both cell populations to maintain their stem cell features and improve their immunosuppressive potential, demonstrated by the inhibition of activated PBMC proliferation in a co-culture setting. As a result, this study suggests articular cartilage derived-CPSC can be used as a novel tool for cellular and acellular regenerative medicine approaches for osteoarthritis (OA). In addition, the benefit of utilizing a biomimetic acellular scaffold as an advanced 3D culture system to more accurately mimic the physiological environment is demonstrated.

scholarly journals Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells

2015 ◽  
Vol 370 (1680) ◽  
pp. 20140365 ◽  
Author(s):  
Maria Rostovskaya ◽  
Nicholas Bredenkamp ◽  
Austin Smith
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Human Pluripotent Stem Cells ◽  
Type I ◽  
Pancreatic Progenitors ◽  
Stem Cell Lines

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification.

Leukemia Stem Cell Potential of Different Progenitor Subpopulations in Myeloid Blast Phase CML

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3489-3489
Author(s):  
Ross Kinstrie ◽  
Dimitris Karamitros ◽  
Nicolas Goardon ◽  
Heather Morrison ◽  
Richard E Clark ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Research Funding ◽  
Cell Populations ◽  
Novel Therapies ◽  
Blast Phase ◽  
Self Renewal ◽  
Stem Cell Populations

Abstract Blast phase (BP)-CML remains the most critical area of unmet clinical need in the management of CML and novel, targeted therapeutic strategies are urgently needed. In the tyrosine kinase inhibitor (TKI) era, the rate of progression to BP is 1 to 1.5% per annum in the first few years after diagnosis, falling sharply when major molecular response is obtained. Around 10% of patients present with de novo BP-CML and despite the use of TKIs, median survival after the diagnosis of BP-CML is between 6.5 and 11 months.Therefore, improved understanding of the biology of BP-CML and novel therapies to prolong therapeutic responses are urgently sought. Studies of myeloid malignancies show that acquisition of tumor-associated mutations occurs principally in a step-wise manner. Initiating mutations usually originate in an hematopoietic stem cell (HSC) to give rise to preleukemic stem cell populations that expand through clonal advantage. Further mutation acquisition and/or epigenetic changes then lead to blast transformation and disruption of the normal immunophenotypic and functional hematopoietic hierarchy. At this stage, multiple leukemic stem cell (LSC) populations (also termed leukemia initiating cell populations) can be identified. We previously showed, in AML, that the CD34+ LSC populations were most closely related to normal progenitor populations, rather than stem cell populations, but had co-opted elements of a normal stem cell expression signature to acquire abnormal self-renewal potential (Goardon et al, Cancer Cell, 2011). CD34+CD38- LSCs were most commonly similar to an early multi-potent progenitor population with lympho-myeloid potential (the lymphoid-primed multi-potential progenitor [LMPP]). In contrast, the CD34+CD38+ LSCs were most closely related to the more restricted granulocyte-macrophage progenitor (GMP). In chronic phase CML, the leukemia-propagating population is the HSC, and the progenitor subpopulations do not have stem cell characteristics. To date, studies to isolate LSC populations in BP-CML have been limited, identifying the GMP subpopulation only as a possible LSC source (Jamieson et al, NEJM, 2004). Furthermore, in vivo LSC activity has not been assessed. We therefore set out to assess the LSC characteristics of different primitive progenitor subpopulations in myeloid BP-CML both in vitro and in vivo. We isolated different stem and progenitor cell subpopulations using FACS; HSC (Lin-CD34+CD38-CD90+ CD45RA-), multipotent progenitor (MPP; Lin-CD34+CD38-CD90-CD45RA-), LMPP (Lin-CD34+CD38-CD90-CD45RA+), common myeloid progenitor (CMP; Lin-CD34+CD38+CD45RA-CD123+), GMP (Lin-CD34+CD38+CD45RA+CD123+) and megakaryocyte erythroid progenitor (MEP; Lin-CD34+CD38+CD45RA-CD123-). The functional potential of these purified populations was examined in 13 patients by: (i) serial CFC replating assays to study progenitor self-renewal (n=10); (ii) In vivo xenograft studies using NSG mice with serial transplantation to identify populations with LSC potential (n=6). Our data conclusively demonstrate that functional LSCs are present in multiple immunophenotypic stem/progenitor subpopulations in myeloid BP-CML, including HSC, MPP, LMPP, CMP and GMP subpopulations. There was inter-patient variability in terms of both in vitro and in vivo functional properties. Fluorescence in situ hybridisation (FISH) was used to assess clonality in the different progenitor subpopulations and identify which populations contained cells with additional cytogenetic abnormalities (ACAs) with a view to improving our understanding of the clonal hierarchy. Interestingly, there were no significant differences in ACAs in the different progenitor subpopulations in the majority of samples studied, suggesting that clonal evolution tends to occur in the HSC compartment in myeloid BP-CML. Preliminary gene expression profiling studies of the different progenitor subpopulations, using Affymetrix Human Gene 1.0 ST Arrays, demonstrated highly variable gene expression, supporting the functional heterogeneity seen. Taken together, our results demonstrate that myeloid BP-CML is a very heterogeneous disorder with variable LSC populations. Further interrogation of these populations will likely identify novel therapies which will specifically target the LSC. Disclosures Copland: Bristol-Myers Squibb: Consultancy, Honoraria, Other, Research Funding; Novartis: Consultancy, Honoraria, Other; Ariad: Consultancy, Honoraria, Research Funding.

In Vitro Electrophysiological Drug Testing Using Human Embryonic Stem Cell Derived Cardiomyocytes

2009 ◽  
Vol 18 (1) ◽  
pp. 161-172 ◽  
Author(s):  
Oren Caspi ◽  
Ilanit Itzhaki ◽  
Izhak Kehat ◽  
Amira Gepstein ◽  
Gil Arbel ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Drug Testing ◽  
Human Embryonic Stem Cell ◽  
Embryonic Stem

Human stem cell-derived encapsulated liver tissue as an effective, consistent and long-lasting in vitro tool for drug testing and development

2017 ◽  
Vol 49 (4) ◽  
pp. e251
Author(s):  
C. Raggi ◽  
M. M’Callum ◽  
C. Mangahas ◽  
Z. Cohen ◽  
A. Shikanov ◽  
...  
Keyword(s):  
Stem Cell ◽  
Liver Tissue ◽  
Drug Testing ◽  
Human Stem Cell

scholarly journals Potency and Fate Specification in CNS Stem Cell Populations In Vitro

2008 ◽  
Vol 3 (6) ◽  
pp. 670-680 ◽  
Author(s):  
Rea Ravin ◽  
Daniel J. Hoeppner ◽  
David M. Munno ◽  
Liran Carmel ◽  
Jim Sullivan ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Populations ◽  
Fate Specification ◽  
Stem Cell Populations

scholarly journals Leptin Receptor As a Functional Marker for Long-Term Repopulating Hematopoietic Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3712-3712
Author(s):  
Thao Trinh ◽  
Scott Cooper ◽  
Arafat Aljoufi ◽  
Edward F. Srour ◽  
Hal E. Broxmeyer
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Functional Marker ◽  
Type I ◽  
Hematopoietic Stem ◽  
Lepr Expression ◽  
Cell Stem Cell

Hematopoietic cell transplantation is an invaluable life-saving regimen for patients affected by malignant and non-malignant hematological disorders. However, successful clinical outcomes depend on the abilities of hematopoietic stem (HSCs) and progenitor cells (HPCs) to home to the bone marrow (BM) and then reconstitute a healthy new blood system. Leptin (Lep), a metabolic hormone well-characterized for its regulations of appetite and body weight by acting on the hypothalamus neurons, has a WSXWS motif of the type I cytokine receptor family and has reported hematopoietic effects (Cioffi et al., Nat Med 1996, Bennett et al., Curr Biol 1996, Umemoto et al., Blood 1997, Gainsford et al. Proc Natl Acad Sci USA 1996, Claycombe et al., Proc Natl Acad Sci USA 2008). These studies were however mostly limited to in vitro assays. Recent work demonstrated that Lep receptor(r)+ stromal cells were indispensable for maintenance of HSC/HPC (Comazzetto et al., Cell Stem Cell 2019, Himburg et al., Cell Stem Cell 2018, Zhou et al., Nat Cell Biol 2017). Yet, whether Lepr expression on HSC/HPC has effects on their in vivo functions remain largely unknown. We hypothesized that environmental factors that affect metabolism of HSCs and HPCs, such as those modulated by Lep/Lepr interactions, may be involved in HSC/HPC regulation and the engraftment of these cells. Using flow cytometry analysis, we first assessed expression levels of Lepr on HSCs and HPCs. While only a low percentage of mouse BM HSC/HPC expressed Lepr, both the percentages of Lepr+HSCs (28.5% Lepr+LT-HSC and 17.2% Lepr+ST-HSC) and mean fluorescence intensity (MFI) of surface Lepr on these cells are significantly higher than that of Lepr+HPCs such as CMP, GMP and CLP (3.8%, 1.5%, 0.7% Lepr+ respectively). Despite the fact that HPCs express a lower level of Lepr, intact Lep/Lepr signaling was critical for their functions. This was illustrated by in vitro colony assay of cells taken from Lepr knockout (-/-) mouse BM in which significantly fewer absolute numbers per femur of HPC-derived colonies (CFU-GM, CFU-GEMM, BFU-E) formed compared to WT controls, and these progenitors were in a slow or non-cycling state. To evaluate how Lepr expression affects in vivo HSC/HPC functions, equal numbers of BM C57BL/6 (WT; CD45.2+) Lepr - Lineage-Sca1+cKit+ (LSK) vs. Lepr+LSK cells were sorted and each transplanted with competitive BoyJ (CD45.1+) cells into lethally irradiated CD45.2+/CD45.1+ F1 recipients. A consistently higher engraftment capacity of Lepr+LSK cells was manifested in comparison to Lepr - LSK cells as noted in peripheral blood (PB) at months 1-6 chimerism post-transplant (91% vs 1.1% at month 6). Lepr+HSCs and Lepr+MPPs expressed similar levels of surface CXCR4 in comparison to corresponding Lepr - populations, suggesting that homing differences may not explain increased engraftment of Lepr+ LSK. At month 6, Lepr+LSK, but not Lepr - cells, demonstrated a significant myeloid-biased engraftment (0.24 vs 0.03 respectively for myeloid/lymphoid ratios). This is consistent with the phenotypic finding that compared to Lepr -LSK cells, Lepr+LSK cells contained a significantly lowered percentage of MPP4 progenitor cells (3.6% vs 36%), which have been demonstrated as a lymphoid-biased subset of MPPs (Pietras et al., Cell Stem Cell 2015). In addition, Lepr+LSK cells contained three-fold fewer progenitors as determined by in vitro colony assays. These findings demonstrated that Lepr+LSK cells were enriched for long-term hematopoietic repopulating HSCs, while its counterpart Lepr -LSK cells contained mostly HPCs. The data also suggested that absence of Lepr expression may play a role in fate-decision skewing HSCs towards MPP4 production. For beginning efforts at mechanistic insight, we hypothesized that Lepr+ HSCs and Lepr+MPP may be different than Lepr - cells in mitochondrial activity. Compared to Lepr - cells, Lepr+HSC and Lepr+MPP cells interestingly possessed more robust mitochondrial metabolism, as demonstrated by their mitochondria having significantly higher membrane potential (measured by JC-1 assay). In summary, Lep/Lepr signaling appears to be a functional ligand-receptor axis for maintaining HSC/HPC homeostasis and differentiation cell bias. Moreover, Lepr expression may serve as a functional marker for long-term repopulating HSCs, which has potential translational possibilities, as Lepr is highly conserved between mice and humans. Disclosures No relevant conflicts of interest to declare.

Bortezomib Has Anti-Proliferative and Apoptotic Effects Against CML Stem Cells, Including the Quiescent Population.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2943-2943 ◽  
Author(s):  
Nicholas Heaney ◽  
Francesca Pellicano ◽  
Lisa Crawford ◽  
Sandra Irvine ◽  
Tessa L. Holyoake
Keyword(s):  
Stem Cell ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Residual Disease ◽  
Therapeutic Window ◽  
Cell Populations ◽  
Wild Type ◽  
Cell Counts ◽  
Apoptotic Effects

Abstract CML is a disease of the haemopoietic stem cell (HSC) caused by the constitutively active tyrosine kinase Bcr-Abl. The current first-line therapy for CML is imatinib (IM), a tyrosine kinase inhibitor (TKI) effective in establishing durable disease control in the majority of patients in chronic phase. However, the quiescent leukaemic HSC (Ph+HSC) population is insensitive to the apoptotic effects of this and more potent TKIs - such as dasatinib or nilotinib. These TKIs exert potent anti-proliferative effects against CML stem and progenitor cells, resulting in accumulation of quiescent cells in vitro. This Ph+HSC reservoir of disease in treated patients is now recognised to be a critical target if we wish to achieve complete eradication of residual disease. Bcr-Abl+ cells have abnormally high levels of proteasome activity and expression and in common with other cancer cells have been shown to be relatively more sensitive to the effects of proteasome inhibitors (PI) than their non-malignant counterparts. We have assessed the potential role for PI in CML using Bcr-Abl+ cell lines (K562 and Ba/F3 with mutant/wild type Bcr-Abl) and Ph+HSC obtained from leukapheresis products of patients, taken with consent, at time of diagnosis with CML. We have demonstrated apoptotic and anti-proliferative effects of PI (MG132 and bortezomib) in wild type and mutated Bcr-Abl+ cell lines (including T315I) and in Ph+HSC (n=7) by viable cell counts, thymidine incorporation and flow cytometry for annexin V and 7AAD. The apoptotic effects also occurred in the quiescent Ph+HSC fraction with an IC50 of 5–7.5nM (bortezomib)- similar to that seen in the total CD34+ cell population and lower than that previously reported for cells from multiple myeloma patients. These results were compared to parallel FACS-sorted cell populations from non-CML Ph- leukapheresis products and a therapeutic window was seen. CFSE tracking of cell division in CML samples in vitro indicated depletion of the quiescent fraction, in contrast to the accumulation of these cells demonstrated with IM, nilotinib or dasatinib, either alone or in combination. The intracellular effects of exposure of these cell populations to bortezomib were assessed by quantitatively measuring the intracellular accumulation of polyubiquitinated proteins seen following PI treatment. Interestingly no clear effect on Bcr-Abl activity was demonstrated with PI treatment of Ph+HSC (measured by immunoblotting with antibody to phosphorylated Crkl), though in Bcr-Abl+ cell lines detectable activity was reduced. Drug combination experiments (Calcusyn) were performed with bortezomib and IM or dasatinib to assess the effect of simultaneous inhibition of Bcr-Abl and the proteasome - the combination of a TKI with a PI resulted in additive effects, however no clear synergistic effects of significance were seen. Our results indicate that PI are capable of inducing CML stem cell specific apoptosis, suggesting the proteasome may be a relevant target for eradication of residual disease in CML following TKI therapy.

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