Putative Stem Cell Population Research Articles

Abstract 5199: Primary culture of human midgut carcinoid cell lines and isolation of a putative carcinoid stem cell population

Abstract Background: Research in carcinoid and neuroendocrine tumors (NETs) of midgut origin is limited by a lack of cell lines. The existing foregut NET lines, BON and H727, are phenotypically distinct from midgut NETs highlighting the importance of establishing midgut derived NET cell lines. Therapeutic options are limited for midgut NETs. NET cell lines are difficult to culture for long periods (thus few cell lines). We are developing methods to improve primary and prolonged NET cell culture in an attempt to identify novel therapeutic approaches. Along these lines, we are conducting studies to identify a putative cancer stem cell population in human NET specimens to provide new insights regarding potential targets in NETs. Methods: Sterile human midgut NET surgical specimens were digested to a single cell suspension and fibroblast depleted with magnetic beads. The NET cells were maintained on collagen I coated plates for primary culture. A portion of the primary cell culture was used for FACS by the Aldefluor assay to isolate potential cancer stem cells (CSCs). Sorted cells were grown in serum-free media on low attachment plates to assess sphere formation and preserved for RNA isolation and subsequent gene array. Subcutaneous xenografts in nude and NOD/SCID gamma mice using fresh tumor implants and NET cell suspensions were performed as potential means to further propagate NET cells. Results: The NET markers, somatostatin receptor 2 and chromogranin A, were detectable in cultures from a midgut carcinoid liver metastasis at passage 3 and 5, as well as in one mouse xenograft. While retention of NET markers from xenograft implants is promising, tumor growth remains limited and slow, with tumors in 5/59 mice over 4-10 months. We are now studying tumor xenografts in more immunodeficient NOD/SCID gamma mice with current patient samples to potentially enhance xenograft growth. FACS sorting identified an ALDH+ population of NET cells, which formed spheres (a characteristic of CSCs) more frequently than ALDH(−) negative cells. A FACS sorted midgut sample plated at 10k cells/well in sphere forming media produced spheres as early as 8 days in the ALDH+ population, while the ALDH- cells did not form spheres (sham sorted cells formed rare spheres), which was also observed in other samples where viable cells were obtained. Additionally, the ALDH+ cells from this population were cutlured on plastic and remained viable for 4 weeks, while the ALDH- population was not viable in culture. RNA isolation for gene array is underway for the ALDH+ population to characterize these cells in midgut carcinoids. Conclusions: We have developed a system allowing for NET primary culture for up to 4 weeks. We have also identified a putative CSC population in NETs. These techniques are the first step in obtaining cells for in vitro studies and in identifying novel therapeutic targets for NETs. Supported by the The Raymond and Beverly Sackler Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5199. doi:10.1158/1538-7445.AM2011-5199

Localization of Label-Retaining Cells in Murine Vocal Fold Epithelium

Epithelial homeostasis is critical for vocal fold health, yet little is known about the cells that support epithelial self-renewal. As a known characteristic of stem cells is that they are slow-cycling in vivo, the purpose of this prospective controlled study was to identify and quantify slow-cycling cells or putative stem cells in murine vocal fold epithelium. Twelve mice were administered daily intraperitoneal injections of a nucleotide dye, bromodeoxyuridine (BrdU), over 7 consecutive days. Under this pulse-chase paradigm, slow-cycling cells retain the dye (label-retaining cells; LRCs) while more rapidly cycling cells lose dye to dilution during multiple cell divisions. The percentage of label-retaining cells (%LRCs) was calculated following a chase period of 2, 4, and 8 weeks postinjections. The %LRCs decreased significantly from 9.4% at 2 weeks to 3.1% at 8 weeks following injections (p < .05). No statistically significant differences in the quantity of BrdU-positive cells were measured between the anterior, mid-membranous, or cartilaginous regions of the vocal fold (p > .05). These findings are consistent with the presence and first report of a small population of putative stem cells along the length of murine vocal fold epithelium.

Sox9 marks adult organ progenitors

Adult stem cells play pivotal roles in self-renewing tissues but also in the post-injury repair of adult organs that normally show little proliferative activity. A new study shows that Sox9 marks a putative adult stem cell population that contributes to the self-renewal and repair of the liver, exocrine pancreas and intestine, three organs of endodermal origin.

Clinical and Functional Relevance of Interactions Between the Bone Marrow Stormal Microenvironment and Primary Human Acute Myeloid Leukemia (AML) Cells

AbstractAbstract 2756 Objectives:Adhesion of acute myeloid leukemia (AML) blasts in the bone marrow (BM) microenvironment confers protection from chemotherapy cytotoxicity. We sought to extend our knowledge of the molecular mechanisms by which the BM stroma support AML by identifying all functionally relevant adhesion receptors that were associated with clinical outcomes, identifying new functional AML-stromal interactions, and examining for aberrant cytokine production by stroma derived from patient AML BM. Methods:We prospectively analyzed by multicolor flow cytometry the expression of 17 different adhesion receptors by AML blasts derived from peripheral blood or BM of 42 adult patients, ages 19–80. Analyses included percent expression, mean fluorescence intensity (MFI), and in 7 patients, the adhesion receptors expressed by the CD34+CD38−CD123+ putative leukemia stem cell (LSC) population. We correlated percent expression and MFI with the likelihood of complete remission (CR) using Wilcoxon two-sample testing with Monte Carlo estimation of the exact p value. Guided by data from the AML blast adhesion receptor analyses, we designed a functional screen to assay primary AML blast adhesion to a normal BM stromal cell line (HS27a). For 10 patients, we assessed the capacity of function blocking monoclonal antibodies directed against a panel of 11 adhesion molecules to inhibit adhesion of purified primary AML blasts to HS27a, allowing functional identification of previously undescribed AML-stromal adhesion determinants not only on AML cells but also on BM stroma. Lastly, we successfully grew primary BM stroma from 5 AML patients, assessed production of 10 cytokines important for normal hematopoiesis, and measured the same cytokines directly in plasma of AML patient BM aspirate specimens. Results:Adhesion receptor analysis demonstrates that the mean expression by all AML patients was >90% for CD11a, β1, PECAM-1, CD44, α4, and α5. Comparison of the adhesion receptor profile of the LSC population to the blast population from which they derived showed a 23.1% +/− 7.7% (mean ± standard error) increase in the expression of α6, and a decrease by 0.4% ± 2.5% for CD34+ blasts compared with all blasts (p = 0.04, Student t-test). We found that in our sample of 18 pretreatment BM samples the 15 patients having CR had a higher MFI for αL (p = 0.021), a higher % blasts expressing α4 (p = 0.039) and PECAM1 (p = 0.012), and a lower MFI for α2 (p = 0.029), α6 (p = 0.028), and CXCR4 (p = 0.005). The data for α4 and CXCR4 agree with prior publications demonstrating improved outcomes for high α4β1 expression and poorer outcomes with high CXCR4 expression (Blood 2009; 113:866–74, J Clin Oncol 2010; 28:2831–8, Blood 2007; 109:786–91). Analyzing the functional importance of adhesion receptors for AML-stromal interactions, we found that function blocking antibodies to β1 (p = 0.0003, Student two-sided t-test), CXCR4 (p < 0.0001), and E-cadherin (p < 0.00002) most strongly inhibited AML blast adhesion to HS27a, with mean inhibitions of 71%, 67%, and 63% respectively (Fig 1). [Display omitted] While β1 and CXCR4 represent known factors in AML-stromal adhesion, we propose a novel role for E-cadherin in AML as a potentially novel therapeutic target whose known biology in other malignancies spans cell-cell adhesion and canonical β-catenin/Wnt signaling. The magnitude of the β1 blocking effect proved consistent with its partnering role with multiple adhesion receptors (e.g. α2, α4, α5, and α6). Analyzing the microenvironment cytokine milieu we found normal BM produced G-CSF, SDF-1, and SCF (6, 3001, and 4857 pg/mL respectively) as expected while some AML specimens produced undetectable levels of G-CSF (4/5 plasmas, 4/5 stromas), SCF (4/5 plasmas), or SDF-1 (2/5 plasmas, 3/5 stromas). Conclusions:Among the 6 receptors correlated with response to induction chemotherapy, high α6 (VLA6 laminin receptor) and CXCR4 MFI correlated with poor outcome. The putative LSC subpopulation showed significantly more α6 expression compared with the entire AML blast population.Antibodies against β1, CXCR4, and E-cadherin exhibited the highest function blocking (60–75% range) of AML-stromal adhesion and our AML-stroma functional assay offers a tool to screen for more novel interactions.Cytokine profiling of the AML microenvironment showed deficiencies in cytokines necessary for normal hematopoiesis which may contribute to the clinical cytopenias in AML. Disclosures:No relevant conflicts of interest to declare.

Canine adipose-derived-mesenchymal stem cells do not lose stem features after a long-term cryopreservation

Adult stem cells are nowadays used for treating several pathologies. A putative stem cell population was found in the adipose tissue of mammals and canine adipose tissue-derived-mesenchymal stem cells (cA-MSC) have been shown to possess the capacity to differentiate into several lineages. The main goal of our research was to fully characterize cA-MSC and examine the effects of cryopreservation on their stemness features. Each sample of cA-MSC was analyzed immediately and then again after being frozen in liquid nitrogen for one year. After the cryopreservation period cells conserved their fibroblast-like morphology, alkaline phosphatase positivity and CD expression but showed a lower proliferation ratio and a lower telomerase activity in comparison with fresh cells. Finally, the cryopreservation protocol did not change the cA-MSC adipogenic, osteogenic and myogenic differentiative potential. Our data demonstrate that stored cA-MSC might represent a promising type of progenitor cell for autologous cellular-based therapies in veterinary medicine.

Identification of epithelial label-retaining cells at the transition between the anal canal and the rectum in mice

In certain regions of the body, transition zones exist where stratified squamous epithelia directly abut against other types of epithelia. Certain transition zones are especially prone to tumorigenesis an example being the anorectal junction, although the reason for this is not known. One possibility is that the abrupt transition of the simple columnar epithelium of the colon to the stratified squamous epithelium of the proximal portion of the anal canal may contain a unique stem cell niche. We investigated whether the anorectal region contained cells with stem cell properties relative to the adjacent epithelium. We utilized a tetracycline-regulatable histone H2B-GFP transgenic mice model, previously used to identify hair follicle stem cells, to fluorescently label slow-cycling anal epithelial cells (e.g. prospective stem cells) in combination with a panel of putative stem cell markers. We identified a population of long-term GFP label-retaining cells concentrated at the junction between the anal canal and the rectum. These cells are BrdU-retaining cells and expressed the stem cell marker CD34. Moreover, tracking the fate of the anal label-retaining cells in vivo revealed that the slow-cycling cells only gave rise to progeny of the anal epithelium. In conclusion, we identified a unique population of cells at the anorectal junction which can be separated from the other basal anal epithelial cells based upon the expression of the stem cell marker CD34 and integrin a6, and thus represent a putative anal stem cell population.

Differential Distribution of erbB Receptors in Human Glioblastoma Multiforme: Expression of erbB3 in CD133-Positive Putative Cancer Stem Cells

Glioblastomas are the most common primary central nervous system tumors in adults, and they remain resistant to current treatments. erbB1 signaling is frequently altered in glioblastomas, suggesting thaterbB receptor family members may represent targets for molecular therapy. We performed a comprehensive analysis of erbB receptor and ligand expression profiles in a panel of 9 glioblastomas andcompared them to nonneoplastic cerebral tissue containing neocortex and adjacent white matter. Quantitative reverse transcription-polymerase chain reaction and Western blot analysis showed that erbB1signaling and erbB2 receptors exhibited highly variable deregulation profiles in the tumors, with patterns ranging from underexpression to overexpression; in contrast, erbB3 and erbB4 were downregulated. We next performed immunohistochemistry to determinethe distribution patterns of erbB receptors among the main neuralcell types in the tumors with special reference to the putative tumor stem cell population. Results revealed intertumoral and intratumoral heterogeneity in all 4 erbB expression profiles, but each receptor exhibited a distinct distribution pattern among glial fibrillary acidic protein-, Olig2-, NeuN-, and CD133-positive populations. Although erbB1 immunoreactivity was detected in only small subsets of CD133-positive putative tumor stem cells, erbB3 immunoreactivity was prominent in this population, suggesting that erbB3 may represent a new potential therapeutic target.

β-catenin Activity in the Dermal Papilla Regulates Morphogenesis and Regeneration of Hair

The activity of keratinocytes in the hair follicle is regulated by signals from a specialized mesenchymal niche, the dermal papilla (DP). Here, mice expressing cre recombinase in the DP were developed to probe the interaction between follicular keratinocytes and the DP in vivo. Inactivation of the beta-catenin gene within DP of fully developed hair follicles results in dramatically reduced proliferation of the progenitors and their progeny that generate the hair shaft, and, subsequently, premature induction of the destructive phase of the hair cycle. It also prevents regeneration of the cycling follicle from stem cells. Gene expression analysis reveals that beta-catenin activity in the DP regulates signaling pathways, including FGF and IGF, that can mediate the DP's inductive effects. This study reveals a signaling loop that employs Wnt/beta-catenin signaling in both epithelial progenitor cells and their mesenchymal niche to govern and coordinate the interactions between these compartments to guide hair morphogenesis.

Complex display of putative tumor stem cell markers in the NCI60 tumor cell line panel.

Tumor stem cells or cancer initiating cells (CICs) are single tumor cells that can regenerate a tumor or a metastasis. The identification and isolation of CICs remain challenging, and a variety of putative CIC markers have been described. We hypothesized that cell lines of the NCI60 panel contain CICs and express putative CIC markers. We investigated expression of putative CIC surface markers (CD15, CD24, CD44, CD133, CD166, CD326, PgP) and the activity of aldehyde dehydrogenase in the NCI60 panel singly and in combination by six-color fluorescence-activated cell sorting analysis. All investigated markers were expressed in cell lines of the NCI60 panel. Expression levels of individual markers varied widely across the 60 cell lines, and neither single marker expression nor simple combinations nor co-expression patterns correlated with the colony-formation capacity of cell lines. Rather, marker expression patterns correlated with tumor types in multidimensional analysis. Whereas some expression patterns correlated with tumor entities such as basal breast cancer, other expression patterns occurred across different tumor types and largely related to expression of a more mesenchymal phenotype in individual breast, lung, renal, and melanoma cell lines. Our data for the first time demonstrate that tumor cell lines display CIC markers in a complex pattern that relates to the tumor type. The complexity and tumor type specificity of marker display creates challenges for the application of cell sorting and other approaches to isolation of putative tumor stem cell populations and suggests that therapeutic targeting strategies will need to take this into account.

Persistent circulating human insulin in sheep transplanted in utero with human mesenchymal stem cells

To determine if mesenchymal stem cells (MSC) derived from human fetal pancreatic tissue (pMSC) would engraft and differentiate in sheep pancreas following transplantation in utero. A three-step culture system was established for generating human fetal pMSC. Sheep fetuses were transplanted during the fetal transplant receptivity period with human pMSC and evaluated for in situ and functional engraftment in their pancreas, liver, and bone marrow. Isolation and expansion of adherent cells from the human fetal pancreas yielded a cell population with morphologic and phenotypic characteristics similar to MSC derived from bone marrow. This putative stem cell population could undergo multilineage differentiation in vitro. Three to 27 months after fetal transplantation, the pancreatic engraftment frequency (chimeric index) was 79%, while functional engraftment was noted in 50% of transplanted sheep. Hepatic and marrow engraftment and expression was noted as well. We have established a procedure for isolation of human fetal pMSC that display characteristics similar to bone marrow-derived MSC. In vivo results suggest the pMSC engraft, differentiate, and secrete human insulin from the sheep pancreas.

Adhesion Receptor Expression by Acute Myeloid Leukemia (AML) Bone Marrow Derived or Circulating Blasts and AML Stem Cells: The Key to Overcoming Chemoresistance.

Abstract 2658Poster Board II-634Adhesion of AML blasts within the bone marrow microenvironment confers chemotherapy resistance. CXCR4 plays a primary role in the retention of stem cells within the marrow, and is associated with poor prognosis in AML (Rombouts et al Blood 104:550, 2004, Konoplev et al Cancer 109:1152, 2007; Spoo et al Blood 109:786, 2007). A CXCR4 inhibitor enhanced chemotherapy cytotoxicity for human AML cells on stroma (Zeng et al Mol Cancer Ther 5:3113, 2006). VLA4 expression is also correlated with prognosis in AML, and our group and others have shown that disruption of VLA4 enhances chemotherapy cytotoxicity (Matsunaga et al Nat Med 9:1158, 2003; Becker et al Blood 113:866, 2009). However, given the number of potential ligands and receptors involved in the interaction of leukemia cells within the bone marrow stroma, there are likely other important binding sites. Fundamental to determining mechanisms to overcome drug resistance will be identification of all critical interactions responsible for retention and/or adhesion to the bone marrow stroma. We therefore sought to fully characterize all potential adhesive interactions utilized by AML blasts. We also examined the leukemia stem cell population, the small number of leukemia cells that are able to sustain and perpetuate the leukemia. We have now defined the adhesion receptor expression pattern (18 receptors) for populations of AML blasts, including circulating vs. marrow, and putative stem cell populations as defined by surface markers. Bone marrow and peripheral blood samples have been obtained at presentation for a total of 38 patients with AML. We performed flow cytometry analysis, gating on sequential populations of cells by forward scatter/side scatter, CD45 vs. side scatter, CD34+, or CD34+/CD38-/CD123+ populations. Expression levels were measured by percent of positive cells (%EXPR) and mean fluorescence intensity (MFI). Our analysis of adhesion receptor expression by AML blasts revealed that some receptors were expressed by 85%–92% of blasts, including CD11a, CD29, CD44, CD49d, CD49e, CD31; some were expressed by <5%, including CD49a, CD49c, CD106; and others exhibited moderate expression 18–62%, such as L-selectin, ICAM-1, CXCR4, CD49f. For a given patient, there was no significant correlation between CXCR4 % EXPR or MFI and expression of other receptors, such as CD49d, CD49f, or CD29. However, there was significant positive correlation (by Spearman rank order correlation) between %EXPR on marrow vs. circulating blasts from the same patient for CD29 [p=.01], CD11a (LFA-1) [p=.004], CXCR4 [p=.03], CD31 (PECAM-1) [p=.03], L-selectin [p=.007], CD44 [p=.04], while others were different, including CD49d [p=.41], CD49e [p=.08], and CD29f [p=.17] (all two-sided p values). When comparing the whole CD34+ population to the CD34+CD38-CD123+ population, there was no significant difference in MFI for the receptors CD29, CD44, or CD49f, but the MFI of CD49d and CD49e was significantly lower on the more primitive (stem) cells [p=0.003 for each ]. When we examined the CD34+/CD38-/CD123+ population isolated by sequential CD38 depletion then CD34 selection, and gated on CD123+ cells, we found uniform high percent expression >90% for CD49d, CD49f, CD44, and the percent expression of CD49f was also lower for the primitive cells in some patients. Thus, there are several adhesion receptors expressed at high level that may contribute to biological behavior and chemotherapy resistance. Overcoming chemoresistance in leukemia stem cells may require targeting of multiple receptors in order to achieve a clinical response. Disclosures:Becker:Leukemia and Lymphoma Society: Research Funding.

CD146 Expression in Primary Bone Marrow MSC Progenitor/Stem Cells Is Dependent On Their In Vivo Location.

Abstract 251Primary MSC progenitor/stem cells (MSC-PC/SC) represent only a minute fraction of the bone marrow cells and they give rise to the well-known mesenchymal stromal cells (MSC) in culture. In vivo, MSC-PC/SC are probable constituents of the hematopoietic stem cell niche, thus playing an important role in supporting, maintaining and controlling hematopoiesis. Enrichment of primary MSC progenitors, i.e. CFU-F, has been recently reported based on expression of surface markers such as CD271, CD146, GD2, SSEA4, etc. Based on the expression of CD271 and CD146 on primary MSC (Quirici et al., Exp. Hematol. 2002; Sacchetti et al, Cell, 2007), we have recently identified two BM subpopulations (CD271+/CD146+/ CD45− and CD271+/CD146−/low/ CD45−) that highly enrich for primary MSC-PC/SC (Tormin et al, Blood 2008, 112[11]:843). These two populations contained all assayable CFU-F and both gave rise to typical cultured MSC (expression of standard surface markers, differentiation capacity into adipocytes, osteoblasts, chondrocytes). Interestingly, MSC derived from CD146−/low cells acquired CD146 expression in culture, and we therefore aimed to further investigate whether CD146 expression correlates to functional differences, e.g. stemness, or possibly differences in localization. CD271/CD146 subpopulations were FACS sorted from lineage-depleted BM-MNC. Single cell sorting of CD271/CD146/CD45− cells (n=6) confirmed the results of our prior CFU-F experiments, i.e. high enrichment of CFU-F and multipotency in the two putative stem cell populations. Six-color FACS analysis of primary BM cells showed that both populations coexpressed typical MSC markers (CD90, CD105, PDGFR-β, STRO-1), but not GD2, SSEA4, and endothelial markers. Single cell multiplex PCR on sorted primary MSC-PC/SC showed that both cell populations were negative for CD45, but did express “early” genes (Oct4, Sox2, Nanog), marker genes for the adipogenic lineage (CEBPA, LPIN1) and osteogenesis-related genes (ALPL, Runx2). Gene expression of CD146 correlated to its surface expression with some CD146 bands also detected among the CD146−/low sorted population. Next, we investigated possible differences in localization utilizing confocal microscopy of normal human BM sections. Reticular CD271/CD146 double positive and reticular CD271 single positive cells were identified. Double positive cells were mainly located adjacent to larger vessels and sinusoids but were also found in the marrow space. In contrast, CD271 single positive cells were primarily found in the endosteal space. These cells were furthermore negative for CD45 in contrast to CD45 coexpressing CD271+/low cells in the marrow space. As expected, CD146 single positive endothelial cells were found surrounding larger vessels. Thus, expression of CD146 in CD271+/CD45− cells correlated with localization (primarily endosteal versus primarily perivascular) and we therefore hypothesized that CD146 expression might be regulated by hypoxia levels. To test this, MSC were cultured under normoxic versus hypoxic conditions using deferoxamine mesylate (DFO) to mimic hypoxia. When sorted CD271+/CD146−/low/CD45− cells were cultured in normoxia, CD146 expression was lower compared with cultures initiated with CD271+/CD146+/CD45− cells in the beginning and up to the 1st passage. Thereafter, CD146 expression was comparable. However, when established MSC (CD146+) were cultured in the presence of DFO, CD146 expression was clearly downregulated, and after 7 days about 25% of cells became CD146 negative compared to 3% in normoxic controls. No changes were observed for CD90 and CD271 expression.Taken together, CD271+/CD146+/CD45– and CD271+/CD146–/low/CD45– bone marrow cells are putative primary MSC stem/progenitor populations. Both are highly enriched for primary MSC progenitors, they have comparable functional characteristics as well as comparable surface marker and gene expression profiles. Differences in CD146 expression correlated to localization and are likely to be caused by differences in oxygen levels. We therefore conclude that CD146 expression allows to distinguish the vary rare (0.19 ± 0.09%) primary endosteal niche MSC (CD271+/CD146−/low/CD45−) from vascular niche MSC (CD271+/CD146+/CD45−, 0.31 ± 0.13% of BM cells). Disclosures:No relevant conflicts of interest to declare.

Potential Eradication of Myeloma Stem Cells by Combining ATRA with WNT and HH Signaling Pathways Inhibitors.

Abstract 2839Poster Board II-815Multiple myeloma (MM) remains largely incurable. The existence of the putative myeloma stem cell population may account for drug-resistance and relapse. Previous investigation showed that the CD138-negative (CD138-) MM cell fraction contains the putative MM stem cells. However, little is known about the molecular characteristics of MM stem cells, which makes it difficult to specifically target such cells. In this study, by comparing gene expression profiles (GEP) of CD138+ and CD138- cells isolated from 10 MM cell lines, we discovered that RARα is the top one over-expressed gene in CD138- MM stem cells. RARα has two major isoforms, RARα1 and RARalpha2. Real-time PCR detected significantly higher expression of RARalpha2 but not of RARalpha1 in CD138- MM cells compared with CD138+ cells. We recently reported that increased RARalpha2 expression at diagnosis resulted in a significantly shorter overall-survival (P = 0.003); importantly, ATRA selectively killed RARalpha2-positive myeloma cells, but not RARalpha2-negative cells. These results suggested that ATRA could be used to eradicate specifically RARalpha2-over-expressing MM stem cells. Indeed, we found that ATRA selectively killed CD138- MM stem cells (P < 0.01) from ARP-1 and OCI-MY5 human myeloma cell lines and 5T33 murine myeloma cells but spared the CD138+ tumor cells from these cell lines. WNT and Hedgehog (HH) signaling pathways were activated in CD138- stem cells. To our surprise, ATRA treatment (10-6M) further increased WNT and HH signaling pathways in CD138- myeloma cells, based on increased protein levels of β-catenin and cleaved Shh/Ihh in ATRA-treated cells. Stimulation of these signaling pathways by LiCl (5 mM) and/or SHH(20 μg/mL) partially abrogated ATRA-induced cytotoxities on CD138- MM cells, demonstrating that stimulation of WNT and HH signaling induced partial ATRA-resistance in CD138- cells. A combinational treatment of ATRA (1 μM), a COX-2 inhibitor celecoxib (shown to inhibit WNT signaling, 50 μM) and a HH signaling inhibitor cyclopamine (10 μM) induced synergistic effects on cell death and growth inhibition of CD138- and RARalpha2-over-expressing MM cells in-vitro. In the 5T33 murine myeloma model, the combination of ATRA (20mg/Kg), celecoxib (10mg/Kg) and cyclopamine (20mg/Kg) induced synergistic inhibition of tumor growth in-vivo. Thus, our study provides novel approaches to specifically target MM stem cells. Disclosures:No relevant conflicts of interest to declare.

Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche

The in vitro analysis of intestinal epithelium has been hampered by a lack of suitable culture systems. Here we describe robust long-term methodology for small and large intestinal culture, incorporating an air-liquid interface and underlying stromal elements. These cultures showed prolonged intestinal epithelial expansion as sphere-like organoids with proliferation and multilineage differentiation. The Wnt growth factor family positively regulates proliferation of the intestinal epithelium in vivo. Accordingly, culture growth was inhibited by the Wnt antagonist Dickkopf-1 (Dkk1) and markedly stimulated by a fusion protein between the Wnt agonist R-spondin-1 and immunoglobulin Fc (RSpo1-Fc). Furthermore, treatment with the gamma-secretase inhibitor dibenzazepine and neurogenin-3 overexpression induced goblet cell and enteroendocrine cell differentiation, respectively, consistent with endogenous Notch signaling and lineage plasticity. Epithelial cells derived from both leucine-rich repeat-containing G protein-coupled receptor-5-positive (Lgr5(+)) and B lymphoma moloney murine leukemia virus insertion region homolog-1-positive (Bmi1(+)) lineages, representing putative intestinal stem cell (ISC) populations, were present in vitro and were expanded by treatment with RSpo1-Fc; this increased number of Lgr5(+) cells upon RSpo1-Fc treatment was subsequently confirmed in vivo. Our results indicate successful long-term intestinal culture within a microenvironment accurately recapitulating the Wnt- and Notch-dependent ISC niche.

Cardiac interstitial cells express GATA4 and control dedifferentiation and cell cycle re-entry of adult cardiomyocytes

Interstitial cells of the adult rat heart were characterized with respect to i) expression of cardiac markers of commitment and differentiation, ii) myogenic potential in vitro and iii) ability to modulate cardiomyocyte differentiation state. We demonstrate for the first time that fibroblasts and a proportion of pericytes in the adult rat heart express the transcription factor GATA4. This appears to be a peculiar property of the heart. Fibroblasts that are also derived from the splanchnopleuric mesoderm, such as those of the gut, or fibroblasts of different embryological origin, such as those of skin and skeletal muscle, lack this property. Of note, a nestin+/GATA4+ putative stem cell population is also detected in the adult heart. GATA4+ cardiac interstitial cells do not display myogenic potential in vitro. However, cardiac fibroblasts, but not skin fibroblasts, stimulate dedifferentiation of adult cardiomyocytes and their re-entry into the cell cycle in vitro, as demonstrated by the high number of cardiomyocytes expressing Ki67, phosphorylated histone H3 (H3P) and incorporating 5-bromodeoxiuridine (BrdU) in the co-cultures. In conclusion, cardiac fibroblasts have peculiar expression of myogenic transcription factors, a property that may have an impact for reprogramming these cells to the myogenic differentiation. In addition, they are able to modulate the behavior of adult cardiomyocytes, a property that may be used to promote dedifferentiation and proliferation of cardiac cells in the damaged myocardium.

Effect of Parthenolide on Stem Cells in Childhood Acute Lymphoblastic Leukaemia.

Current therapies for the treatment of childhood acute lymphoblastic leukaemia (ALL) have resulted in vastly improved survival rates of around 80% in recent years. Despite these successes, around 15% of patients die of the disease and relapse is the most common cause of treatment failure. Intensification of treatment to prevent or treat relapse may not be a feasible approach due to an increased risk of significant adverse effects. It is possible that ALL may be maintained by a subpopulation of stem cells that are resistant to regimens designed to kill the bulk population and subsequent relapses may arise from these stem cells. Consequently, there is a need to assess the efficacy of therapeutic agents on ALL stem cells. We have previously shown that ALL cells that are capable of initiating and sustaining the disease in serial xenografts have a CD34+/CD19− phenotype. Furthermore, these putative ALL stem cells were resistant to treatment in vitro with dexamethasone and vincristine, two agents routinely used in the treatment of paediatric leukaemia. In this investigation we have examined the effects of the sesquiterpene lactone parthenolide (PTL), a natural compound which induces oxidative stress and inhibits NF-kB. PTL effectively eradicates stem cells in AML and B-CLL in vitro while sparing normal heamopoietic cells. Unsorted leukaemia cells from 11 cases, with mixed prognostic subgroups, were co-cultured with and without PTL at a range of 0–10μM for 18–24 hours. Cell viability and apoptosis were evaluated by flow cytometry using annexin V and propidium iodide staining. Four out of the 11 cases were relatively resistant to treatment with PTL with only small reductions in viability (<5%) and no significant effect on apoptosis, even at the highest dose evaluated. There was no correlation between the prognostic risk group and the response to PTL. Primary cells from the 4 resistant cases were sorted into CD34+/CD19+ and CD34+/CD19− subfractions to assess the effect of PTL on these cells. The effects of PTL on the CD34+/CD19+ population were similar to that observed with the unsorted leukaemia cells. The CD34+/CD19− population was completely resistant to treatment with PTL, with more cells surviving treatment than the unsorted cells (P=0.03). In the 7 responding cases, the viability of the unsorted cells decreased to 28.4±7.1% and 38±12% were apoptotic following treatment. Very similar effects were observed with the CD34+/CD19+ subfraction in these responding cases with viability reduced to 33.4±6% and 35.9±14% were apoptotic. In contrast, the CD34+/CD19− cells from these 7 cases were significantly resistant to PTL with viabilities >75% at all concentrations evaluated (P<0.003). Apoptosis was 2.6-fold lower at 10μM PTL in the CD34+/CD19− subfraction compared to the unsorted cells (P=0.05). FISH analyses were performed on the viable cells at the end of the time-course and confirmed that leukaemia cells were surviving treatment with PTL. CD34+/CD38− cells from normal peripheral blood samples were also found to be resistant to treatment with PTL and survival of these cells at 10μM was not significantly different to that observed in the CD34+/CD19− ALL population in all 11 cases (P=0.38). Studies to assess the functional capacity of PTL-treated ALL cells are ongoing. These data demonstrate that while PTL shows promising effects on the bulk leukaemia population in some ALL cases, it had no significant effect on the putative ALL stem cell population. In each case examined, the CD34+/CD19− cells were resistant to short term exposure to PTL and responded in a similar manner to normal haemopoietic stem cells. These findings highlight the importance of evaluating therapeutic agents in the context of leukaemia stem cell populations and not just on the bulk leukaemia. Future studies are warranted to gain insight into how the drug sensitivity of ALL stem cells may be mediated.

Quantitative proliferation dynamics and random chromosome segregation of hair follicle stem cells

Regulation of stem cell (SC) proliferation is central to tissue homoeostasis, injury repair, and cancer development. Accumulation of replication errors in SCs is limited by either infrequent division and/or by chromosome sorting to retain preferentially the oldest 'immortal' DNA strand. The frequency of SC divisions and the chromosome-sorting phenomenon are difficult to examine accurately with existing methods. To address this question, we developed a strategy to count divisions of hair follicle (HF) SCs over time, and provide the first quantitative proliferation history of a tissue SC during its normal homoeostasis. We uncovered an unexpectedly high cellular turnover in the SC compartment in one round of activation. Our study provides quantitative data in support of the long-standing infrequent SC division model, and shows that HF SCs do not retain the older DNA strands or sort their chromosome. This new ability to count divisions in vivo has relevance for obtaining basic knowledge of tissue kinetics.

Improved Viability of Random Pattern Skin Flaps through the Use of Adipose-Derived Stem Cells

Flap necrosis caused by inadequate blood supply is a common postoperative complication in reconstructive surgery. Because a putative stem cell population within the adipose tissue has been found to possess angiogenic potential, the authors sought to determine whether these cells might selectively induce neovascularization and increase the viability of random pattern skin flaps. Adipose-derived stem cells were isolated from the inguinal fat pads of ICR mice and expanded ex vivo for three passages. After the elevation of cranially based random pattern skin flaps (3 cm long and 1 cm wide), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI)-labeled adipose-derived stem cells were then injected into the pedicle base (group A) or 1.5 cm distal to the pedicle (group B). Medium containing no adipose-derived stem cells, mature adipocytes, or basic fibroblast growth factor were injected in three other control groups separately (n = 10 for each group). Millimetric measurements were taken at postoperative day 7 for evaluation of flap viability. Specimens were harvested for histologic analyses. Adipose-derived stem cells led to a statistically significant increase in flap viability in both group A and group B compared with the control and the adipocyte groups. Histologic examination also demonstrated a statistically significant increase in capillary density in both group A and group B. Moreover, some of the endothelial cells were stained positively for DiI. These findings suggest that adipose-derived stem cells have a potential for enhancing the blood supply of random pattern skin flaps. This mechanism might be both the direct differentiation of adipose-derived stem cells into endothelial cells and the indirect effect of angiogenic growth factor released from adipose-derived stem cells.

Enrichment of putative stem cells from adipose tissue using dielectrophoretic field-flow fractionation.

We have applied the microfluidic cell separation method of dielectrophoretic field-flow fractionation (DEP-FFF) to the enrichment of a putative stem cell population from an enzyme-digested adipose tissue derived cell suspension. A DEP-FFF separator device was constructed using a novel microfluidic-microelectronic hybrid flex-circuit fabrication approach that is scaleable and anticipates future low-cost volume manufacturing. We report the separation of a nucleated cell fraction from cell debris and the bulk of the erythrocyte population, with the relatively rare (<2% starting concentration) NG2-positive cell population (pericytes and/or putative progenitor cells) being enriched up to 14-fold. This work demonstrates a potential clinical application for DEP-FFF and further establishes the utility of the method for achieving label-free fractionation of cell subpopulations.

Androgen receptor expression in prostate cancer stem cells: is there a conundrum?

Androgen deprivation therapy (ADT) is standard frontline therapy for metastatic prostate cancer. However, prostate cancer progresses to a castrate-resistant state. The response of prostate cancer to androgen deprivation is mediated by the androgen receptor (AR). Castrate-resistant disease is marked by a gain-of-function in AR and AR reactivation. The stem cell hypothesis of cancer would therefore predict that AR should be expressed in the prostate cancer stem cell, since genetic selection for gain-of-function changes in AR, such as AR gene amplification, should occur at the level of the stem cell population. Initial reports characterizing prostate cancer stem cells suggest that AR is not expressed in this population, which is an apparent conundrum. Here, we examined the CD44+/24- LNCaP putative stem cell population by in-cell Western and show that AR is expressed at the protein level. Our findings suggest that at least a subset of prostate cancers express AR in the putative stem cell population.