scholarly journals Development of a Patient-Derived Xenograft Model of Human Myelodysplastic Syndromes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5217-5217
Author(s):  
Maria Krevvata ◽  
Cedric Dos Santos ◽  
Xiaochuan Shan ◽  
Anthony Secreto ◽  
Gwenn Danet-Desnoyers ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Myelodysplastic Syndromes ◽  
Bone Marrow Failure ◽  
Hematopoietic Cells ◽  
Xenograft Model ◽  
Bone Marrow Aspiration ◽  
Initial Assessment ◽  
Refractory Anemia

Abstract Background: Myelodysplastic syndromes (MDS) comprise a group of hematologic disorders characterized by ineffective hematopoiesis and cytopenias that may lead to acute myeloid leukemia (AML) or bone marrow failure. Although a few genetic mouse models have been generated, they cannot recapitulate the heterogeneity of the disease. The attempts for the development of a xenograft model have been challenging in the past, mainly due to poor engraftment of MDS hematopoietic cells. Recent scientific evidence pinpoint the importance of the niche in the establishment and progression of hematological malignancies and suggested a novel approach to establishing MDS xenotransplantation models. In this study we aim to develop a mouse xenotransplantation model of human MDS and to evaluate the role of mesenchymal stem cells (MSCs) in the engraftment process. Methods: MSC's from normal donors and MDS patient samples were generated and characterized using standard culture methods. NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSGS) mice were used as recipients. Mice were injected with 1 x 106 bone marrow mononuclear cells (MNC) alone or in combination with 5 x 105 MSC's. MSC's were from normal donors, from same patient as MNC's (autologous) or from other MDS patients (MDS allogeneic). All cells were injected intrafemorally. Mice were assessed for engraftment by bone marrow aspiration in the ipsilateral bone at 6 weeks, in the contralateral bone at 10 weeks and at necropsy at 12-22 weeks (depending on initial assessment). Long-term engrafted samples were further assessed for presence of stem cells by secondary transplantation into NSGS mice. 1 x 106 human CD45 selected hematopoietic cells were passaged again without or with 5 x 105 MSC's. Secondary recipients were assessed at 12 weeks for engrafment. MDS samples represented both high risk (refractory anemia with excess blasts, RAEB)) and low risk (refractory anemia (RA), refractory cytopenia with myelodysplasia (RCMD)) disease. The overall degree of engraftment was assessed by bone marrow aspiration and measurement of human CD45+ cells. Lymphoid, myeloid and erythroid engraftment was assessed. Results: Five out of five injected MDS samples showed persistent human cells when assessed at 6 weeks. For most samples, engraftment at 6 weeks was low (<2%) and was not consistently influences by the presence or absence of MSC. On week 10, only two out of five patients had increased engraftment and only one showed higher engraftment levels in the mice co-transplanted with MSCs. Both of these better engrafting samples were from patients with RAEB. Engrafting samples demonstrated both myeloid and erythroid engraftment. Molecular analysis to confirm engraftment of MDS clone will be presented. In total, 2 out of 5 patients showed long-term engraftment (> 12 weeks). One of these samples has been transferred to secondary animals. Secondary transplanted mice injected with selected hCD45+ cells with or without MSC's showed variable engraftment levels on week 10 after injections, with the majority of those not reaching long-term engraftment. Conclusions: Passive transferof MDS hematopoietic cells as assessed at 6 weeks after intrabone injection is highly consistent and not dependent on MSC when total MNC fraction is used. However, long-term engraftment of MDS cells in xenotransplanted mice is uncommon suggesting that true MDS stem cells do not consistently engraft. Further analysis with injection into different mouse strains with purified CD34+ cells is under way. Disclosures Dos Santos: Amgen: Employment.

Engraftment of Human MDS Samples in Xenotransplanted Mice Depends on Human Cytokines but Not Mesenchymal Stem Cells (MSC)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1980-1980
Author(s):  
Maria Krevvata ◽  
Cedric Dos Santos ◽  
Xiaochuan Shan ◽  
Selene Nunez-Cruz ◽  
Anthony Secreto ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Research Funding ◽  
Bone Marrow Aspiration ◽  
Initial Assessment ◽  
Refractory Anemia ◽  
Nsg Mice ◽  
Human Cytokines

Abstract Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases with limited treatment options. Development of a patient derived xenotransplantation model for pre-clinical studies is a priority goal in the field. It has been hypothesized that patient derived mesenchymal stem cells and/or human cytokines are necessary to establish MDS engraftment in immunocompromised mice. In this study we evaluated the ability of mesenchymal stem cells (MSC) to facilitate the engraftment process having as a goal to generate a robust patient-derived xenograft (PDX) mouse model for MDS. We also assessed the contribution of human cytokines as expressed in transgenic mice. Methods: MSC from normal donors and MDS patient samples were generated and characterized using standard culture methods. NOD.Cg-Prkdcscid
Il2rgtm1Wjl/SzJ (NSG) and NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSG-S) mice were used as recipients. Mice were injected with 1 x 106 bone marrow mononuclear cells (MNC) alone or in combination with 5 x 105 MSC. MSC were from normal donors, from same patient as MNC (autologous), from other MDS patients or normal donors (allogeneic). All cells were injected intrafemorally. Mice were assessed for engraftment by bone marrow aspiration at 6-8, 10 weeks and at necropsy at 12-32 weeks (depending on initial assessment). Long-term engrafted samples were further assessed for presence of stem cells by secondary transplantation into NSG-S mice. 1 x 106 human CD45+ cells were passaged without or with 5 x 105 MSC. Secondary recipients were assessed at 12 weeks for engrafment. MDS samples represented both high risk (refractory anemia with excess blasts, RAEB)) and low risk (refractory anemia (RA), refractory cytopenia with myelodysplasia (RCMD), chronic myelomonocytic leukemia (CMML)) disease. The overall degree of engraftment was assessed by bone marrow aspiration and measurement of human CD45+ cells by means of FACS. Lymphoid, myeloid and erythroid engraftment was assessed similarly. 5 x 105 MSCs from normal donors were transduced with GFP (pELNS.CBR-T2A-GFP) and intrafemorally injected into NSG mice and the presence of human MSC was evaluated by in vivo Imaging (Xenogen Spectrum system and Living Image Version 4.3 software.). Results: 12 out of 12 injected MDS samples showed persistent human cells when assessed at 6-8 weeks. For most samples, engraftment at that time-point was low (<2%) and was not consistently influenced by the presence or absence of MSC. On week 10, 4 out of 12 injected MDS patient samples showed increased engraftment and only one showed higher engraftment levels in the mice co-transplanted with MSC. Two of these were from patients with RAEB and CMML and showed both myeloid and erythroid engraftment. Molecular analysis showed consistent engraftment of the malignant clone in 2 out of 2 patient samples tested. Overall, only 3 out of 12 patients showed long-term engraftment (> 12 weeks) and these samples were transferred to secondary animals. Secondary transplanted mice injected with selected hCD45+ cells with or without MSC showed variable engraftment levels on week 10 after injections. One of them reached long-term engraftment (>12 weeks), whereas the second gradually lost engraftment after week 10 and the third is ongoing. MSC tracking showed a gradual loss of MSC after intrafemoral injections to the point that no MSC were detected 3 weeks later. Comparison of engraftment in NSG mice to NSG-S mice (the latter producing human cytokines) showed a slight increase in engraftment at week 6 which continued at later time points. Conclusions: Passive transfer of MDS hematopoietic cells as assessed at 6-8 weeks after intrafemoral injection is highly consistent and not dependent on MSC when total MNC fraction is used. The latter may be explained since MSC are lost within the first three weeks after injections. Direct comparison of NSG vs. NSG-S mice showed that the presence of hGM-CSF, hIL-3 and h-SCF in the NSG-S mice increased the engraftment levels. Successful long-term engraftment of MDS cells in xenotransplanted mice was achieved for one patient sample so far, indicating that MDS initiating cells can be maintained in NSG-S mice. However, establishing long-term engraftment can only be achieved with a subset of patient samples. MDS engraftment is influenced by human cytokines but not with MSC cells which may suggest that MDS does not depend on MSC's for long term growth. Disclosures Nunez-Cruz: Novartis: Research Funding. Milone:Novartis: Patents & Royalties, Research Funding.

Mesenchymal Stem Cells from Patients with Myelodysplastic Syndromes Are Functionally and Genomically Abnormal.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1916-1916
Author(s):  
Olga López Villar ◽  
Fermin M. Sánchez-Guijo ◽  
Juan Luis García ◽  
Jose Ramon González Porras ◽  
Eva Villarón ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Myelodysplastic Syndromes ◽  
Array Cgh ◽  
Hematopoietic Cells ◽  
Refractory Anemia ◽  
Comparative Genomic ◽  
Hematopoietic Stem ◽  
Lower Expression

Abstract Myelodysplastic syndromes (MDS) are a group of clonal disorders of hematopoietic stem cell (HSC). The hematopoietic microenvironment plays a major role in the physiology of the hematopoietic system, and mesenchymal stem cells (MSC) are not only the progenitors but also the key regulators of this microenviroment. Recently, some data has been published showing that these MSC could be involved in the MDS pathophysiology. Moreover, the presence of cytogenetic aberrations on these cells is controversial. The aim of the study was to characterize bone marrow derived MSC from patients with MDS using different approaches: kinetic studies, immunophenotypic analysis and genetic changes by array-based comparative genomic hybridization (array-CGH). FISH was performed with the probe of 1q31 and Q-PCR was performed with the SYBR Green technique in order to confirm array-CGH results. Patients with untreated MDS were included in the study. Median age was 72 years (range: 54–89). Diagnosis of MDS was established according to the WHO classification as follows: 5q- syndrome (n=7), refractory anemia (n=2), refractory anemia with ringed sideroblasts (n=1) and refractory anemia with excess blasts type 2 (n=3). Standard cytogenetic and FISH studies on hematopoietic cells were performed at diagnosis according to standard methods. MSC from MDS were compared to those from 12 healthy donors. MSC were obtained by plating mononuclear cells from bone marrow, and cultured and expanded following standard procedures. According to the international consensus for MSC characterization, in the third passage MSC were harvested to perform phenotypical studies and cytogenetics. To perform Array-CGH a total of 3500 genomic targets were compounded from RP-11 libraries. The PCR products after purification were arrayed onto glass slides using a BioRobot. DNA was labelled, denaturalised and hybridizated. MSC from MDS achieved confluence at a slower rate than donor-MSC [23 days (range 12–90) vs 15 days (range 11–30) p&lt;0,01]. Also some phenotypical markers showed lower expression on patients MSC: CD105 and CD104 (p&lt;0,05%), compared to MSC from bone marrow donors. In all MDS cases analysed MSC showed DNA genomic changes. The most frequent aberrations were 1q31q32 region gains, present in 72% of cases, and 5q31 loss in 46% of patients. Gains in 1q31 were confirmed by FISH using the probe obtained from the BAC. Loss of 17p13 occurred in 3 cases (28%), and this may be relevant since p53 is included in that region, Q-PCR was subsequently performed confirming the loss of p53 in all these cases. The changes were not observed in hematopoietic cells analysed in order to exclude somatic changes. We conclude that MSC from MDS are functionally abnormal since they show a slower growing capacity and a lower expression of adhesion molecules. In the present study it is shown for the first time that MSC from MDS show several genomic aberrations when CGH arrays are used and the data have been confirmed by FISH and Q-PCR.

Exhaustion of Donor Hematopoietic Stem Cells in Lethally-Irradiated Hosts Can Be Sbstantially Mitigated by Targeting p18INK4C.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1200-1200
Author(s):  
Hui Yu ◽  
Youzhong Yuan ◽  
Xianmin Song ◽  
Feng Xu ◽  
Hongmei Shen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Kinase Inhibitor ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Total Period ◽  
Over Expression

Abstract Hematopoietic stem cells (HSCs) are significantly restricted in their ability to regenerate themselves in the irradiated hosts and this exhausting effect appears to be accelerated in the absence of the cyclin-dependent kinase inhibitor (CKI), p21. Our recent study demonstrated that unlike p21 absence, deletion of the distinct CKI, p18 results in a strikingly positive effect on long-term engraftment owing to increased self-renewing divisions in vivo (Yuan et al, 2004). To test the extent to which enhanced self-renewal in the absence of p18 can persist over a prolonged period of time, we first performed the classical serial bone marrow transfer (sBMT). The activities of hematopoietic cells from p18−/− cell transplanted mice were significantly higher than those from p18+/+ cell transplanted mice during the serial transplantation. To our expectation, there was no detectable donor p18+/+ HSC progeny in the majority (4/6) of recipients after three rounds of sBMT. However, we observed significant engraftment levels (66.7% on average) of p18-null progeny in all recipients (7/7) within a total period of 22 months. In addition, in follow-up with our previous study involving the use of competitive bone marrow transplantation (cBMT), we found that p18−/− HSCs during the 3rd cycle of cBMT in an extended long-term period of 30 months were still comparable to the freshly isolated p18+/+ cells from 8 week-old young mice. Based on these two independent assays and the widely-held assumption of 1-10/105 HSC frequency in normal unmanipulated marrow, we estimated that p18−/− HSCs had more than 50–500 times more regenerative potential than p18+/+ HSCs, at the cellular age that is equal to a mouse life span. Interestingly, p18 absence was able to significantly loosen the accelerated exhaustion of hematopoietic repopulation caused by p21 deficiency as examined in the p18/p21 double mutant cells with the cBMT model. This data directly indicates the opposite effect of these two molecules on HSC durability. To define whether p18 absence may override the regulatory mechanisms that maintain the HSC pool size within the normal range, we performed the transplantation with 80 highly purified HSCs (CD34-KLS) and then determined how many competitive reconstitution units (CRUs) were regenerated in the primary recipients by conducting secondary transplantation with limiting dilution analysis. While 14 times more CRUs were regenerated in the primary recipients transplanted with p18−/−HSCs than those transplanted with p18+/+ HSCs, the level was not beyond that found in normal non-transplanted mice. Therefore, the expansion of HSCs in the absence of p18 is still subject to some inhibitory regulation, perhaps exerted by the HSC niches in vivo. Such a result was similar to the effect of over-expression of the transcription factor, HoxB4 in hematopoietic cells. However, to our surprise, the p18 mRNA level was not significantly altered by over-expression of HoxB4 in Lin-Sca-1+ cells as assessed by real time PCR (n=4), thereby suggesting a HoxB4-independent transcriptional regulation on p18 in HSCs. Taken together, our current results shed light on strategies aimed at sustaining the durability of therapeutically transplanted HSCs for a lifetime treatment. It also offers a rationale for the feasibility study intended to temporarily target p18 during the early engraftment for therapeutic purposes.

Suppression of Cyclin D 1 (CD1) by on 01910.Na Is Associated with Decreased Survival or Trisomy 8 Myelodysplastic Bone Marrow: A Potential Targetted Therapy for Trisomy 8 MDS.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1651-1651
Author(s):  
Aarthi Shenoy ◽  
Loretta Pfannes ◽  
Francois Wilhelm ◽  
Manoj Maniar ◽  
Neal Young ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cyclin D1 ◽  
Cultured Cells ◽  
Bone Marrow Failure ◽  
Refractory Anemia ◽  
Trisomy 8 ◽  
Monosomy 7 ◽  
Diploid Cells

Abstract CD34 positive cells from patients with trisomy 8 myelodysplastic syndrome (MDS) have pronounced expression of early apoptotic markers compared to normal hematopoietic cells. However, trisomy 8 clones persist in patients with bone marrow failure and expand following immunosuppression (Sloand EM et al; Blood2005; 106(3):841). We have demonstrated up-regulation of c-myc, survivin, and CD1 in CD34 cells of patients with trisomy 8 (Sloand et al; Blood2007; 109(6):2399). Employing siRNA mediated knockdown of the anti-apoptotic protein survivin, we demonstrated a decrease in trisomy 8 cell growth and postulated that increased Cyclin D1 caused the upregulation of survivin resulting in resistance of these cells to apoptosis. Using fluorescent in situ hybridization (FISH) we showed that the novel styryl sulfone, ON 01910.Na (Vedula MS et al; European Journal of Medicinal Chemistry2003;38:811), inhibits cyclin D1 accumulation and is selectively toxic to trisomy 8 cells while promoting maturation of diploid cells. Flow cytometry of cultured cells demonstrated increased proportions of mature CD15 positive myeloid cells and decreased number of immature CD33+ cells or CD34+ blasts (Sloand EM et al; Blood2007;110:822). These encouraging in vitro data led to a phase I/II trial of ON 01910.Na in MDS patients with refractory anemia with excess blasts who had IPSS =/&gt; int-2. This study was designed to assess the safety, and activity of escalating doses of ON 01910.Na (800 mg/m2/day × 3 days, 800 mg/m2/day × 5 days, 1500 mg/m2/day × 5 days, 1800 mg/m2/day × 5 days every 2 weeks) in MDS patients. To date five MDS patients have been treated with ON 01910.Na for 4 to 16 weeks in the first two dose cohorts. Two patients had isolated trisomy 8, two had complex cytogenetic abnormalities including trisomy 8 in all aneuploid cells, and one had monosomy 7. Three and five day infusions were well tolerated. Pharmakokinetic analysis showed that the half life of the drug is 1.3 ± 0.5 hours without signs of drug accumulation. Four of five patients demonstrated a rapid and significant decrease in the number of peripheral blasts and aneuploid cells after 4 weeks of therapy (see below), concomitantly with increases in neutrophil and/or platelet counts in four patients. All four patients exhibiting a biological effect of drug treatment had trisomy 8 in their aneuploid clone prior to therapy. One monosomy 7 patient, previously refractory to EPO became responsive to Darbopoietin and another trisomy 8 patient became platelet-transfusion independent. In this early safety trial, ON 01910.Na demonstrates efficacy at early timepoints with respect to improved cytopenias and decreased blast counts. Continued enrollment and long term follow-up will further detail clinical efficacy and impact on the long term prognosis of high risk MDS patients treat with this drug. Figure Figure

scholarly journals Retroviral vector design for long-term expression in murine hematopoietic cells in vivo

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1812-1822 ◽  
Author(s):  
PH Correll ◽  
S Colilla ◽  
S Karlsson
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Detrimental Effect ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Copy Numbers ◽  
Viral Copy Number ◽  
Gc Gene

Abstract A series of retroviral vectors containing the human glucocerebrosidase (GC) cDNA driven by various promoters have been constructed in an attempt to discover which vector design can most efficiently transduce murine hematopoietic stem cells (HSCs) and drive expression of the transferred gene in hematopoietic cells of mice reconstituted with the transduced stem cells. The simplest vector, LG, in which the GC gene is driven by the viral LTR, was the most efficient vector at infecting HSCs, with an average viral copy number in hematopoietic tissues of 3 copies/cell in recipient mice. In general, the viral vectors that contained any additional promoters or enhancers to drive expression of either the GC gene or a selectable marker gene (Neo) had lower titers and/or transduced HSCs at a lower efficiency. This was seen most markedly when the human phosphoglycerate (PGK) promoter was used to drive the human GC cDNA. Despite repeated attempts to obtain a high titer producer clone, this virus consistently produced low titers and subsequently resulted in the lowest proviral copy numbers in long-term reconstituted mice. Only the viral LTR and PGK promoter were capable of driving significant levels of human GC RNA in hematopoietic cells of long-term reconstituted mice, with a much lower level of RNA generated by an internal herpes TK or SV40 immediate early promoter. Insertion of the internal transcription unit in the opposite orientation relative to the viral LTRs had a detrimental effect on gene expression. The levels of RNA generated by a hybrid LTR containing the myeloproliferative sarcoma virus enhancer were higher in bone marrow-derived macrophages than in nonadherent cells of the bone marrow when compared with the LG vector. The presence of an internal promoter to drive expression of the human GC cDNA did not seem to have a detrimental effect on expression levels from the viral LTR. In fact, in the presence of an internal TK or PGK promoter expression from the LTR was increased despite the presence of lower proviral copy numbers. Insertion of a second gene (Neo) into the vector had a negative impact on long-term expression in hematopoietic cells in vivo; however, this seems to be due solely to the lower transduction efficiency of this vector. Overall, the highest levels of GC activity in macrophages of long-term reconstituted mice were generated by the LG vector; however, these levels were variable.(ABSTRACT TRUNCATED AT 400 WORDS)

Hypoxia-reoxygenation induces premature senescence in FA bone marrow hematopoietic cells

Blood ◽  
2005 ◽  
Vol 106 (1) ◽  
pp. 75-85 ◽  
Author(s):  
Xiaoling Zhang ◽  
June Li ◽  
Daniel P. Sejas ◽  
Qishen Pang
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Cells ◽  
Premature Senescence ◽  
Wild Type ◽  
Cell Cycle Profile ◽  
Cycle Arrest ◽  
Associated Proteins

Hematopoietic cells are often exposed to transient hypoxia and reoxygenation as they develop and migrate. Given that bone marrow (BM) failure occurred in patients with Fanconi anemia (FA), we reason that hypoxia-then-reoxygenation represents a physiologically relevant stress for FA hematopoietic progenitor/stem cells. Here we show that expansion of Fancc–/– BM cells enriched for progenitor and stem cells was significantly decreased after 2 continuous cycles of hyperoxic-hypoxic-hyperoxic treatments compared with wild-type (WT) BM cells. This inhibition was attributable to a marked decrease of lineage-depleted (Lin–) ScaI– c-kit+ cells and more primitive Lin– ScaI+ c-kit+ cells in Fancc–/– BM cells following reoxygenation. Evaluation of the cell-cycle profile of long-term BM culture (LTBMC) revealed that a vast majority (70.6%) of reoxygenated Fancc–/– LTBMC cells was residing in the G0 and G1 phases compared with 55.8% in WT LTBMC cells. Fancc–/– LTBMC cells stained intensely for SA-β-galactosidase activity, a biomarker for senescence; this was associated with increased expression of senescence-associated proteins p53 and p21WAF1/CIP1. Taken together, these results suggest that reoxygenation induces premature senescence in Fancc–/– BM hematopoietic cells by signaling through p53, up-regulating p21, and causing senescent cell-cycle arrest. Thus, reoxygenation-induced premature senescence may be a novel mechanism underlying hematopoietic cell depletion and BM failure in FA.

TNFα Overproduction in FANCC-Deficient Cells Is TLR8 Dependent.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 494-494
Author(s):  
Scott Vanderwerf ◽  
Johanna Svahn ◽  
Praveen Anur ◽  
Ricardo Pasquini ◽  
Grover C. Bagby
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Clonal Evolution ◽  
Hematopoietic Cells ◽  
Mononuclear Phagocytes ◽  
C Cells ◽  
Mutant Cells

Abstract Abstract 494 The Fanconi anemia (FA) proteins play a role in regulating genome stability but it is not clear that loss of genoprotection in FA hematopoietic cells accounts for the molecular pathogenesis of bone marrow failure so characteristic of this disease. Other factors are known to influence survival and replication of FA stem cells. For example, not only are FA progenitors and stem cells hypersensitive to the apoptotic effects of TNFα, FA cells over-produce TNFα. Most importantly over-production of and hypersensitivity to TNFα in hematopoietic cells of Fancc-/- mice results in bone marrow hypoplasia 1;2 and long-term ex-vivo exposure of murine Fancc -/- hematopoietic cells to both growth factors and TNFα results in the evolution of cytogenetically marked preleukemic clones.3 Therefore, the hematopoietic phenotype of FA is likely multifactorial and may evolve from the overproduction of precisely the cytokine to which FA stem cells are hypersensitive. Methods: We sought to clarify the molecular basis of aberrant TNFα-production. We conducted gene expression microarray experiments using RNA samples from low density marrow cells obtained from 11 normal volunteers and 22 Fanconi anemia patients with uncomplicated marrow hypoplasia without clonal cytogenetic defects. Because the FA complex is known to enhance ubiquitinylation of FANCD2, we reasoned that the ubiquitinylation state of proteins involved in the TNF pathways might also be influenced by core FA proteins. Therefore, we conducted in vitro ubiquitinylation assays using hexahistidine-tagged ubiquitin and an ATP-recycling system added to lysates of FANCC-deficient lymphoblasts (HSC536) and control cells (isogenic cells complemented with WT FANCC cDNA). Following the ubiquitinylation reaction, ubiquitinylated proteins were affinity purified, digested and analyzed by 2D capillary LC-MS/MS. Mass spectra were obtained and peptide precursor-MS/MS spectrum pairs were analyzed using SEQUEST and support vector machine learning.4 Peptides identified only in one or the other cell line were considered. Results: Initially we anticipated focusing on the set of proteins uniquely ubiquitinated in normal cells. However, the transcriptomal results indicated that genes encoding proteins in the ubiquitin pathway were over-represented in the list of genes that were over-expressed in FA samples. Consequently, we examined both differential ubiquitination lists and found that a major regulator of TNF-gene expression, TLR8, appeared in the ubiquitinylated fraction only in mutant cells. In co-immunoprecipitation studies we confirmed that TLR8 (or a TLR8-associated protein) is ubiquitinylated in mutant FA-C cells, and using RNAi determined that high level TNFα synthesis in mutant cells depended upon TLR8 and its downstream signaling intermediates IRAK-1 and IKK-alpha/beta. FANCC deficient THP1 blue cells were created using lentiviral shRNA targeting FANCC. These cells exhibited the MMC hypersensitive phenotype and over-expressed both TNFα and an NF-kappaB reporter gene (secreted embryonic alkaline phosphatase) in response to TLR8 agonists but not to other TLR agonists. Primary splenic macrophages from Fancc-/- mice were also hypersensitive to the TLR8 agonist R848. TNFα production in FA-C cells was suppressed by inhibitors of TLR8, p38 MAPK, IRAK, and IKK. Engineered point mutants of FANCC were capable of complementing the mitomycin C hypersensitivity phenotype of FANCC mutant cells but did not suppress TNFα overproduction in FANCC mutant cells. In conclusion, TNF over-expression in FANCC-deficient cells reflects the loss of FANCC function as a suppressor of TLR8 activation. In addition, FANCC suppresses TLR8 dependent production of TNFα in normal mononuclear phagocytes at least in part by suppressing either TLR8 ubiquitinylation or by inhibiting its association with an ubiquitinylated protein. Finally, this function of FANCC is independent of its function in protecting the genome from cross-linking agent-induced damage. In light of the role of TNFα in bone marrow failure and clonal evolution in this disease, control of TNF-production by targeting the TLR8 pathway might provide an opportunity to enhance hematopoietic activity and forestall clonal evolution in patients with this disorder. 1. Sejas DP, et al, J Immunol 2007;178:5277-5287. 2. Zhang × et al, J.Cell Sci. 2007;120:1572-1583. 3. Li J, et al, J.Clin.Invest. 2007;117:3283-3295, 4. Anderson DC, et al, J Proteome.Res 2003;2:137-146. Disclosures: No relevant conflicts of interest to declare.

Significant Engraftment of Immature Hematopoietic Cells From Patients with Low Risk Myelodysplastic Syndromes (MDS) in Immunodeficient Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1694-1694
Author(s):  
Hind Medyouf ◽  
Florian Nolte ◽  
Maximilian Mossner ◽  
Verena Nowak ◽  
Bettina Zens ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mesenchymal Stromal Cells ◽  
Myelodysplastic Syndromes ◽  
Stromal Cells ◽  
Hematopoietic Cells ◽  
Low Risk ◽  
Cell Phenotype

Abstract Abstract 1694 Introduction: Myelodysplastic syndromes are a heterogeneous group of malignant clonal hematologic disorders characterized by ineffective hematopoiesis, peripheral cytopenias and dysplastic bone marrow cells, with frequent progression to acute myeloid leukemia. Because of its heterogeneous nature, modeling of this disease has proven to be very difficult in cell culture systems as well as mice. In addition, attempts to generate a xenotransplant model in immuno-compromised mice have only achieved very low levels of engraftment that are often transient, making it very difficult to study the biology of this disease in vivo. Recent studies in mice have shown that conditional impairment of the small RNA processing enzyme Dicer in mouse osteolineages induced a stromal niche that promoted myelodysplasia, leading to the hypothesis that abnormal bone marrow stromal cells might provide a “fertile soil“ for the expansion of the malignant clone. Patients and Methods: To the date of writing, a total of 12 primary hematopoietic stem cell- and mesenchymal stroma cell (MSCs) samples selected from patients with MDS have been isolated and xenotransplanted into NOD.Cg-Prkdscid Il2rgtm1Wjl/Szj (NSG) mice: MDS 5q- (n=7), MDS RCMD (n=3), MDS RAEB I (n=1), MDS-U (n=1). Engraftment was monitored by FACS using human specific antibodies to CD45, CD34 and CD38. In addition cell cycle behavior was analyzed by Ki67/Hoechst staining. Mesenchymal stromal cells were characterized using previously described stromal markers: CD105, CD271, CD73, CD166, CD90, CD146 and CD44. To isolate genomic DNA and RNA for molecular analyses, MDS xenografts were flow sorted based on human CD45 expression. Molecular characterization of primary MDS samples and xenotransplants was carried out by serial copy number analysis using Affymetrix SNP 6.0 Arrays, metaphase cytogenetics and direct sequencing of known mutations in the transplanted MDS samples. Results: We show, that the concomitant transplantation of MDS-derived mesenchymal stromal cells with the corresponding hematopoietic patient stem/progenitor cells leads to significant and long-term engraftment (0.1 – 15% for up to 23 weeks) of cells isolated from IPSS low and intermediate risk MDS patients. In addition to the bone marrow, MDS hematopoietic cells also infiltrate other hematopoietic compartments of the mouse including the spleen. Significant engraftment of cells with progenitor (CD34+CD38+) as well as stem cell phenotype (CD34+CD38-) was observed, which is consistent with engraftment of an MDS stem cell that sustains long-term hematopoiesis. SNP array analysis confirmed the clonal origin of the engrafted cells as MDS xenografts harboring the identical genomic lesions as present in the patient disease. Conclusion: We present a robust MDS xenograft model of low risk MDS entities based on the concomitant transplantation of primary MDS hematopoietic cells with MSCs from the same patients. This model does not only allow to study the biology of this disease in vivo but also the molecular and cellular interactions between MSCs and hematopoietic MDS cells. In addition it provides a useful platform to study the effects of new experimental therapeutic agents for the treatment of MDS in molecularly defined MDS cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Human embryonic stem cell–derived hematopoietic cells are capable of engrafting primary as well as secondary fetal sheep recipients

Blood ◽  
2006 ◽  
Vol 107 (5) ◽  
pp. 2180-2183 ◽  
Author(s):  
A. Daisy Narayan ◽  
Jessica L. Chase ◽  
Rachel L. Lewis ◽  
Xinghui Tian ◽  
Dan S. Kaufman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Xenograft Model ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Hematopoietic Activity

The human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and postnatal human sources. CD34+/lineage- or CD34+/CD38- cells isolated from human embryonic stem cells (hESCs) differentiated on S17 feeder layer were transplanted by intraperitoneal injections into fetal sheep. Chimerism in primary transplants was established with polymerase chain reaction (PCR) and flow cytometry of bone marrow and peripheral blood samples. Whole bone marrow cells harvested from a primary recipient were transplanted into a secondary recipient. Chimerism was established as described before. This animal was stimulated with human GM-CSF, and an increase in human hematopoietic activity was noted by flow cytometry. Bone marrow aspirations cultured in methylcellulose generated colonies identified by PCR to be of human origin. We therefore conclude that hESCs are capable of generating hematopoietic cells that engraft primary recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the secondary recipient.

scholarly journals Retroviral vector design for long-term expression in murine hematopoietic cells in vivo

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1812-1822 ◽  
Author(s):  
PH Correll ◽  
S Colilla ◽  
S Karlsson
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Detrimental Effect ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Copy Numbers ◽  
Viral Copy Number ◽  
Gc Gene

A series of retroviral vectors containing the human glucocerebrosidase (GC) cDNA driven by various promoters have been constructed in an attempt to discover which vector design can most efficiently transduce murine hematopoietic stem cells (HSCs) and drive expression of the transferred gene in hematopoietic cells of mice reconstituted with the transduced stem cells. The simplest vector, LG, in which the GC gene is driven by the viral LTR, was the most efficient vector at infecting HSCs, with an average viral copy number in hematopoietic tissues of 3 copies/cell in recipient mice. In general, the viral vectors that contained any additional promoters or enhancers to drive expression of either the GC gene or a selectable marker gene (Neo) had lower titers and/or transduced HSCs at a lower efficiency. This was seen most markedly when the human phosphoglycerate (PGK) promoter was used to drive the human GC cDNA. Despite repeated attempts to obtain a high titer producer clone, this virus consistently produced low titers and subsequently resulted in the lowest proviral copy numbers in long-term reconstituted mice. Only the viral LTR and PGK promoter were capable of driving significant levels of human GC RNA in hematopoietic cells of long-term reconstituted mice, with a much lower level of RNA generated by an internal herpes TK or SV40 immediate early promoter. Insertion of the internal transcription unit in the opposite orientation relative to the viral LTRs had a detrimental effect on gene expression. The levels of RNA generated by a hybrid LTR containing the myeloproliferative sarcoma virus enhancer were higher in bone marrow-derived macrophages than in nonadherent cells of the bone marrow when compared with the LG vector. The presence of an internal promoter to drive expression of the human GC cDNA did not seem to have a detrimental effect on expression levels from the viral LTR. In fact, in the presence of an internal TK or PGK promoter expression from the LTR was increased despite the presence of lower proviral copy numbers. Insertion of a second gene (Neo) into the vector had a negative impact on long-term expression in hematopoietic cells in vivo; however, this seems to be due solely to the lower transduction efficiency of this vector. Overall, the highest levels of GC activity in macrophages of long-term reconstituted mice were generated by the LG vector; however, these levels were variable.(ABSTRACT TRUNCATED AT 400 WORDS)

Sign in / Sign up

Export Citation Format

Share Document