Molecular Epidemiological Investigation of Human Parainfluenza 3 Virus Outbreak in a Pediatric Bone Marrow Transplantation Unit

Abstract Background Human parainfluenza virus 3 (hPIV3), a common cause of respiratory infections in children, can cause nosocomial outbreaks in patients undergoing hematopoietic stem cell transplantation, resulting in significant morbidity and mortality. Between July and August 2016, an increased number of hPIV3 infections were noted in a pediatric bone marrow transplant unit (BMT). Two patients were identified in late July and 4 patients in August. We undertook molecular typing of hPIV3 to determine whether cases represented multiple introductions of community virus strains or patient to patient transmission of a single strain. Previous reports of molecular typing have targeted either the F (fusion protein) gene or HN (hemagglutinin-neuraminidase) gene. We compared results using both methods direct from clinical specimens. Methods Nasopharyngeal (NP) swabs from 6 patients in BMT ward and 6 patients hospitalized on other wards had hPIV3 detected by the Luminex NxTAG Respiratory Pathogen Panel over 2 months. For the F gene a single pair of primers were used to first amplify then sequence a 278 basepair (bp) region by reverse-transcriptase PCR (RT-PCR). For HN gene a 1719 bp region was amplified using nested RT-PCR, then sequenced with 6 sets of overlapping primers. The resulting contigs were assembled manually with ContigExpress. Phylogenetic analysis of assembled sequences was performed in MEGA7 using the maximum likelihood method. Results For the HN gene sequence of 1715 bp was obtained for 10 of 12 patients (5 in each group). Phylogenetic analysis of HN sequences indicated 2 distinct hPIV3 lineages (Figure 1). The 5 BMT patients differed by a maximum of 1bp, while 5 samples from other wards differed by 14 to 57 bp. For the F gene only 98 bp of common sequence was obtained for 7 patients, all of whom had HN gene sequences available. Phylogenetic analysis of F gene sequence also supported the presence of 2 distinct lineages. Conclusion Molecular typing of hPIV3 suggests there was transmission of a single hPIV3 strain within the BMT unit despite protective isolation of all BMT patients in positive pressure single rooms and the use of contact and droplet precautions for infected cases. We found sequencing the HN gene more informative than sequencing the F gene. Disclosures All authors: No reported disclosures.

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Engraftment of Human Mesenchymal Stem Cells upon Transplantation into Newborn Rag2−/−gc−/− Mice.

Blood ◽

10.1182/blood.v108.11.1652.1652 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1652-1652

Author(s):

Patrick Ziegler ◽

Steffen Boettcher ◽

Hildegard Keppeler ◽

Bettina Kirchner ◽

Markus G. Manz

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cord Blood ◽

Human Mesenchymal Stem Cells ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Gm Csf ◽

Cd34 Cells ◽

Gene Transcripts

Abstract We recently demonstrated human T cell, B cell, dendritic cell, and natural interferon producing cell development and consecutive formation of primary and secondary lymphoid organs in Rag2−/−gc−/− mice, transplanted as newborns intra-hepatically (i.h.) with human CD34+ cord blood cells (Traggiai et al., Science 2004). Although these mice support high levels of human cell engraftment and continuous T and B cell formation as well as CD34+ cell maintenance in bone marrow over at least six month, the frequency of secondary recipient reconstituting human hematopoietic stem and progenitor cells within the CD34+ pool declines over time. Also, although some human immune responses are detectable upon vaccination with tetanus toxoid, or infection with human lymphotropic viruses such as EBV and HIV, these responses are somewhat weak compared to primary human responses, and are inconsistent in frequency. Thus, some factors sustaining human hematopoietic stem cells in bone marrow and immune responses in lymphoid tissues are either missing in the mouse environment, or are not cross-reactive on human cells. Human mesenchymal stem cells (MSCs) replicate as undifferentiated cells and are capable to differentiate to multiple mesenchymal tissues such as bone, cartilage, fat, muscle, tendon, as well as marrow and lymphoid organ stroma cells, at least in vitro (e.g. Pittenger et al., Science 1999). Moreover, it was shown that MSCs maintain CD34+ cells to some extend in vitro, and engraft at low frequency upon transplantation into adult immunodeficient mice or fetal sheep as detected by gene transcripts. We thus postulated that co-transplantation of cord blood CD34+ cells and MSCs into newborn mice might lead to engraftment of both cell types, and to provision of factors supporting CD34+ maintenance and immune system function. MSCs were isolated and expanded by plastic adherence in IMDM, supplemented with FCS and cortisone (first 3 weeks) from adult bone marrow, cord blood, and umbilical vein. To test their potential to support hemato-lymphopoiesis, MSCs were analyzed for human hemato-lymphotropic cytokine transcription and production by RT-PCR and ELISA, respectively. MSCs from all sources expressed gene-transcripts for IL-6, IL-7, IL-11, IL-15, SCF, TPO, FLT3L, M-CSF, GM-CSF, LIF, and SDF-1. Consistently, respective cytokines were detected in supernatants at the following, declining levels (pg/ml): IL-6 (10000-10E6) > SDF-1 > IL-11 > M-CSF > IL-7 > LIF > SCF > GM-CSF (0–450), while FLT3L and TPO were not detectable by ELISA. Upon i.h. transplantation of same passage MSCs (1X10E6) into sublethally irradiated (2x2 Gy) newborn Rag2−/−gc−/− mice, 2-week engraftment was demonstrated by species specific b2m-RT-PCR in thymus, spleen, lung, liver and heart in n=7 and additionally in thymus in n=3 out of 13 animals analyzed. Equally, GFP-RNA transcripts were detectable in the thymus for up to 6 weeks, the longest time followed, upon co-transplantation of same source CD34+ cells and retrovirally GFP-transduced MSCs in n=2 out of 4 animals. Further engraftment analysis of ongoing experiments will be presented. Overall, these results demonstrate that human MSC produce hemato-lymphoid cytokines and engraft in newborn transplanted Rag2−/−gc−/− mice, at least at early time-points analyzed. This model thus might allow studying hematopoietic cell and MSC-derived cell interaction, and might serve as a testing system for MSC delivered gene therapy in vivo.

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Notch Signaling Pathway in Hematopoietic Stem Cells Is Activated through Interactive Communication with Mesenchymal Stem Cells.

Blood ◽

10.1182/blood.v110.11.1408.1408 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1408-1408

Author(s):

Yuji Kikuchi ◽

Akihiro Kume ◽

Masashi Urabe ◽

Hiroaki Mizukami ◽

Takahiro Suzuki ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Notch Signaling ◽

Notch Signaling Pathway ◽

Cell Contact ◽

Rt Pcr ◽

Interactive Communication ◽

Hematopoietic Stem ◽

Notch Ligands

Abstract Mesenchymal stem cells (MSCs), which are key elements of hematopoietic microenvironment in bone marrow, are known to play a critical role in supporting hematopoiesis. A variety of hematopoietic growth factors are produced from MSCs, and cell-to-cell contact is also believed to be crucial in the interaction between hematopoietic stem cells (HSCs) and MSCs. However, the molecular mechanisms of hematopoiesis-supporting ability of MSCs are still unclear. In particular, there is little information regarding the effects of HSCs on MSC function. In the present study, we investigated the cellular and molecular events in the interactive communication between HSCs and MSCs using a differentiation-inducible MSC model; i.e. parent C3H10T1/2 cells and 10T1/2-derived cell lines, A54 preadipocytes and M1601 myoblasts. These cells were co-cultured with murine HSCs (Lin-Sca1+) isolated from bone marrow. There was 9-fold increase in the number of hematopoietic progenitors after co-culture with A54 preadipocytes, whereas there was no increase when co-cultured with parent 10T1/2 or M1601 cells. More intriguingly, cobblestone areas were observed only when HSCs were co-cultured with A54 cells. Quantitative RT-PCR showed that A54 cells express significantly higher levels of SCF, SDF-1, and angiopoietin-1 (Ang-1) compared with parent 10T1/2 cells and M1601 cells, although these cytokines were not up-regulated when co-cultured with HSCs. To search for the genes involved in the interaction between HSCs and MSCs, we compared gene expression profiles before and after the co-culture by using a microarray analysis. Among the candidate genes with up-regulation after the co-culture, we paid attention to the Notch system, because Notch ligands are considered to play an important role in nurturing HSCs within the hematopoietic microenvironment. As a result, the expression of Notch ligands, Jagged1 and Dll3, increased in A54 cells after the coculture with HSCs. On the other hand, the expression of Notch1 and Hes-1 also increased in HSCs upon co-culture with A54 cells. These data were confirmed by quantitative RT-PCR. Moreover, when HSCs were co-cultured with A54 cells without cell-to-cell contact using Transwell permeable supports, there was neither increase in the number of progenitors in the upper wells, nor the up-regulation of Notch ligands in A54 cells in the lower wells. These findings support the idea that HSCs act on MSCs to induce the expression of Notch ligands via direct cell-to-cell contact and that the Notch ligands derived from MSCs act on HSCs in turn to activate Notch signaling pathway, possibly leading to the cobblestone formation with the maintenance of immature state of HSCs. The Notch system may be one of the critical elements in the interactive communication between HSCs and MSCs.

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The Biological Characteristics of Osteoblasts Derived from Myelodysplastic Syndrome Patients.

Blood ◽

10.1182/blood.v110.11.4101.4101 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4101-4101

Author(s):

Wen-ming Chen ◽

Zi-xing Chen ◽

Jian-nong Cen ◽

Jun He ◽

Xiao-li Jiao ◽

...

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndrome ◽

Biological Characteristics ◽

Bone Marrow Mononuclear Cell ◽

Feeder Layer ◽

Rt Pcr ◽

Osteoblastic Cell Line ◽

Hematopoietic Stem ◽

Gm Csf

Abstract It was hypothesized that osteoblasts play a central role in hematopoiesis, and it has been shown that osteoblasts produce many factors essential for the survival, renewal, and maturation of hematopoietic stem cells (HSCs). By using human fetal osteoblastic cell line hFOB1.19 as a model of control, we investigated the biological characteristics of osteoblasts derived from patients with myelodysplastic syndrome (MDS) and their hematopoietic supportive function in vitro. MSCs isolated from bone marrow of MDS patients and normal donors were cultured and checked for their morphology, immunophenotype, CFU-F forming capacity and the expression of hematopoietic cytokines. A feeder layer was prepared by osteoblasts induced from 3rd generation of cultured MSCs and treated with mitomycin C. Ficoll-isolated bone marrow mononuclear cell from normal donors were then seeded on the feeder layer to co-culture in vitro without exogenous cytokines. FCM revealed that both MSCs and hFOB cells were positive for CD44, CD73(SH3), CD105(SH2) and CD90 (Thy1), but negative for CD34, CD45, HLA-DR. RT-PCR found that hFOB cells expressed mRNA of SCF, IL-6, IL-11, SDF-1, GM-CSF and G-CSF. MSCs obtained from MDS patients and normal donors were displaying fibroblastoid morphology. Their growth pattern, immunophenotype and CFU-F forming capacity were similar (P >0.05). Without exogenous cytokines, the osteoblasts derived from MDS could sustain GM-CFC survival for at least 3 weeks. The CFU-GM yield from cells in upper layer of co-culture was not different from those of control in hematopoiesis supportive experiments in vitro (P>0.05). RT-PCR clearly demonstrated that the cultured BM-MSCs from normal donor expressed mRNA of SCF, SDF-1, IL-6, and IL-11. As the MSCs differentiated toward osteoblasts, the expression of G-CSF could be detected, whereas GM-CSF remained undetectable. The same expression profile of above cytokines were also seen in osteoblasts induced from BM-MSCs of MDS patients. In conclusion, osteoblasts may play a pivotal role in hematopoiesis. The biological characteristics of osteoblasts from bone marrow of MDS patients were generally not different from those of osteoblasts in bone marrow of normal controls. Both of them could support survival of GM-CFC hematopoietic progenitor cells in vitro, according to their expression of multiple cytokines. These findings suggested that the osteoblasts derived from MDS patients may not be involved in the malignant process.

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Partial Characterization and In Vitro Expansion of Putative CLL Precursor/Stem Cells Which Are Dependent on Bone Marrow Microenvironment for Survival

Blood ◽

10.1182/blood.v116.21.2433.2433 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2433-2433

Author(s):

Medhat Shehata ◽

Rainer Hubmann ◽

Martin Hilgarth ◽

Susanne Schnabl ◽

Dita Demirtas ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Healthy Persons

Abstract Abstract 2433 Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of B lymphocytes which typically express CD19 and CD5. The disease remains incurable and recurrence often occurs after current standard therapies due to residual disease or probably due to the presence of therapy-resistant CLL precursors. Based on the growing evidence for the existence of leukemia stem cells, this study was designed to search for putative CLL precursors/stem cells based on the co-expression of CLL cell markers (CD19/CD5) with the hematopoietic stem cell marker (CD34). Forty seven CLL patients and 17 healthy persons were enrolled in the study. Twenty four patients had no previous treatment and 23 had pre-therapy. Twenty two patients were in Binet stage C and 25 patients in B. Twenty two patients had unmutated and 18 mutated IgVH gene (7: ND). Cytogenetic analysis by FISH showed that 14 patients had del 13q, 8 had del 11q, 4 had del 17p and 9 had trisomy 12. Peripheral blood and bone marrow mononuclear cells were subjected to multi-colour FACS analysis using anti-human antibodies against CD34, CD19 and CD5 surface antigens. The results revealed the presence of triple positive CD34+/CD19+/CD5+ cells in CLL samples (mean 0.13%; range 0.01–0.41) and in healthy donors (0.31%; range 0.02–0.6) within the CD19+ B cells. However, due to the high leukocyte count in CLL patients, the absolute number of these cells was significantly higher in CLL samples (mean: 78.7; range 2.5–295 cells /μL blood) compared to healthy persons (mean: 0.45: range 0.04–2.5 cells/μl)(p<0,001). These triple positive “putative CLL stem cells” (PCLLSC) co-express CD133 (67%), CD38 (87%), CD127 (52%), CD10 (49%), CD20 (61%), CD23 (96%), CD44 (98%) and CD49d (74%). FISH analysis on 4 patients with documented chromosomal abnormalities detected the corresponding chromosomal aberrations of the mature clone in the sorted CD34+/CD5+/CD19+ and/or CD34+/CD19-/CD5- cells but not in the CD3+ T cells. Multiplex RT-PCR analysis using IgVH family specific primer sets confirmed the clonality of these cells. Morphologically, PCLLSC appeared larger than lymphocytes with narrow cytoplasm and showed polarity and motility in co-culture with human bone marrow stromal cells. Using our co-culture microenvironment model (Shehata et al, Blood 2010), sorted cell fractions (A: CD34+/19+/5+, B: CD34+/19-/5- or C: CD34-/CD19+/5+) from 4 patients were co-cultured with primary autologous human stromal cells. PCLLSC could be expanded in the co-culture to more than 90% purity from fraction A and B but not from fraction C. These cells remained in close contact or migrated through the stromal cells. PCLLSC required the contact with stromal cells for survival and died within 1–3 days in suspension culture suggesting their dependence on bone marrow microenvironment or stem cell niches. RT-PCR demonstrated that these cells belong to the established CLL clone. They also eexpress Pax5, IL-7R, Notch1, Notch2 and PTEN mRNA which are known to play a key role in the early stages of B cells development and might be relevant to the early development of the malignant clone in CLL. Using NOD/SCID/IL2R-gamma-null (NOG) xenogeneic mouse system we co-transplanted CLL cells from 3 patients (5 million PBMC/mouse) together with autologous bone marrow stromal cells (Ratio: 10:1). The percentage of PCLLSC in the transplanted PBMC was 0.18% (range 0.06–0.34%). Using human-specific antibodies, human CD45+ cells were detected in peripharal blood of the mice (mean 0.9 % range 0.47–1.63%) after 2 months of transplantation. More than 90% of the human cells were positive for CD45 and CD5. Among this population, 26% (range 15–35%) of the cells co-expressed CD45, CD19, CD5 and CD34 and thus correspond to the PCLLSC. In conclusion, our data suggest the existence of putative CLL precursors/stem cells which reside within the CD34+ hematopoietic stem cell compartment and carry the chromosomal aberrations of the established CLL clone. These cells could be expanded in vitro in a bone marrow stroma-dependent manner and could be engrafted and significantly enriched in vivo in NOG xenotransplant system. Further characterization and selective targeting and eradication of these cells may pave the way for designing curative therapeutic strategies for CLL. Disclosures: No relevant conflicts of interest to declare.

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Real-Time RT-PCR Analysis of Individual Osteolineage Cells within the Hematopoietic Stem Cell Niche

Blood ◽

10.1182/blood.v118.21.2389.2389 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2389-2389

Author(s):

Lev Silberstein ◽

Masatake Osawa ◽

Charles Lin ◽

Peter Kharchenko ◽

Cristina Lo Celso ◽

...

Keyword(s):

Bone Marrow ◽

Pcr Analysis ◽

Rna Seq ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Chi Squared ◽

The Individual ◽

Osteolineage Cells ◽

Angiopoietin 1

Abstract Abstract 2389 Osteolineage cells (OLCs) have been shown to participate in a regulatory bone marrow microenvironment for the hematopoietic stem and progenitor cells (HSPCs) – the endosteal niche. Our previous experiments using live animal imaging have demonstrated that single transplanted HSPCs preferentially home in close proximity to the individual OLCs. We hypothesized that these HSPC-proximal cells represent a distinct subpopulation of OLCs, which is specifically involved in a non-cell autonomous regulation of HSPC quiescence and self-renewal. To test this hypothesis, we developed a novel experimental platform, which allows visualization of HSPC-OLC cell pairs in-vivo and retrieval of the individual OLCs for molecular analysis. We intravenously injected DiI labeled adult bone marrow-derived FACS-sorted Lin−Sca1+c-kit+CD34−Flk2− HSPCs into irradiated newborn collagen 2.3GFP mouse recipients; in this transgenic strain, the majority of the OLCs are labeled with green fluorescent protein (GFP). 48 hours later, we sacrificed the animals and obtained fresh unfixed sections of femoral trabecular bone. Using a combination of differential interference contrast fluorescent microscopy, in-situ enzymatic digestion and micromanipulation, we harvested individual GFP-positive OLCs located within 2 cell diameters (“niche” OLCs) or greater than 5 cell diameters (“control” OLCs) from single DiI-bright HSPCs. Following reverse transcription and cDNA amplification with 29 cycles of PCR, as per the single cell RNA-Seq protocol (Tang et al, Nature Protocols 2010), we performed real-time RT-PCR analysis of 31 samples – 15 niche cells and 16 controls - for the OLC signature genes (osteocalcin, osterix) and for the genes implicated in playing a functional role in the HSPC-OLC cell interaction (osteopontin, CXCL12, angiopoietin 1). Transcripts for GAPDH, collagen 1 and GFP served as positive controls for the amplification. As expected, all cells were positive for GFP and over 85% cells expressed collagen 1. Osteopontin and CXCL12 were expressed at a similar level and frequency in the niche and control OLCs. However, we found that angiopoietin 1 transcripts were detected exclusively in the niche OLCs (3/15 versus 0/16, p <0.05 by Chi-squared). Moreover, niche OLCs were enriched for the osterix-positive cells (7/15 versus 2/16, p <0.05 by Chi-squared) and expressed a lower level of osteocalcin, as normalized for GAPDH expression (1.13 vs. 0.97, p< 0.05 by t-test). Our results suggest that niche OLCs may have a distinct molecular signature and reside within a population of very immature OLCs, as evidenced by the osterix + osteocalcin low phenotype. Further unbiased transcriptome characterization of these cells using genome-wide RNA-Seq assay is therefore likely to provide more evidence in support of our hypothesis and reveal novel non-cell autonomous regulators of HSPC quiescence. To our knowledge, this approach represents the first attempt to define molecular heterogeneity in-vivo at a single cell level using the micro-anatomical relationship between two heterologous cell types. Disclosures: Scadden: Fate Therapeutics: Equity Ownership.

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Hybrid AAV6/2-Mediated Human β-Globin Expression in β-Thalassemia Patient Hematopoietic Stem Cells Transplanted Into Irradiated BALB/c Nude Mouse

Blood ◽

10.1182/blood.v118.21.4715.4715 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4715-4715

Author(s):

Mengqun Tan ◽

Zhenqing Liu ◽

Juan Zhang ◽

Zhiyan Li ◽

Liujiang Song ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Bone Marrow ◽

Globin Gene ◽

Blood Transfusions ◽

Rt Pcr ◽

Thalassemia Patient ◽

Hematopoietic Stem ◽

Human Hscs

Abstract Abstract 4715 β -Thalassemia is one of the most common worldwide monogenic human diseases,caused by molecular defects in the human β -globin gene cluster leading to decrease or absence of β-globin. Loss of β -globin chains causes ineffective production of oxygen-carrying hemoglobin and therefore results in severe anemia. The treatment for β -Thalassemia major usually includes lifelong blood transfusions but chronic blood transfusion often causes iron overload, and accumulated iron produces tissue damage in multiple organs, so that iron chelating treatment is also needed. Bone marrow transplantation is another effective therapy, which can eliminate a patient's dependence on blood transfusions, however, it is difficult to find a matching donor for most patients; therefore it is only available for a minority of patients. Gene therapy is one potential novel therapy for treatment of inherited monogenic disorders. The long–term therapeutic strategy for this disease is to replace the defective β-globin gene via introduction of a functional gene into hematopoietic stem cells (HSCs). Adeno-associated virus type 2 (AAV), a nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. AAV can infect both dividing and non-dividing cells and wild AAV integrates preferentially at a specific site on human chromosome 19. In the absence of helper virus, recombinant AAV will stably integrate into the host cell genome, mediating long-term and stable expression of the transgene. In this study, we used a hybrid rAAV6/2 vector carrying the human β-globin gene to transduce HSCs from a β -Thalassemia patient, followed by transplantation into irradiated BALB/c nude mice. One month post-transplantation, Hb was prepared from peripheral blood and analyzed by Western Blot and HPLC respectively. RNA and DNA were isolated from bone marrow cells (BMCs) from recipient mice transplanted with mock-infected or hybrid rAAV–globin-infected cells and analyzed by RT-PCR and PCR respectively. The results showed: 1. Human β-actin and β-globin transcripts were detected by RT-PCR in BMCs from all recipient mice, indicating that human HSCs were successfully transplanted in these mice and that the human β-globin gene was transcriptionally active in the donor cells. 2. The level of human hemoglobin expressed in peripheral red blood cells of recipient mice as measured by HPLC (ratio of β/α) was increased to 0.3 from 0.05 of pre-transplantation levels. Expression of human β-globin was also confirmed in recipient mice by Western Blot; a 2–3-fold increase compared with that of controls. Our results indicate that human HSCs from a β-Thalassemia patient can be efficiently transduced by a hybrid rAAV6/2-β-globin vector followed by expression of normal human β-globin protein. This study provides a proof-of-concept that rAAV6/2-mediated gene transfer into human HSCs might be a potential approach for gene therapy of β-Thalassemia. Disclosures: No relevant conflicts of interest to declare.

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Recombinant scAAV2 Vector-Mediated Ex Vivo Transduction of Primary Human Hematopoietic Stem Cells from a β-Thalassemia Patient and Human β-Globin Gene Expression in a Murine Xenograft Model in Vivo

Blood ◽

10.1182/blood.v124.21.5951.5951 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5951-5951

Author(s):

Liu-Jiang Song ◽

Xin-Hua Zhang ◽

Jun Zhu ◽

Jue-Lian Wu ◽

Xiao-Ling Yin ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Globin Gene ◽

Rt Pcr ◽

Post Transplantation ◽

Hematopoietic Stem ◽

Human Hscs ◽

Globin Gene Expression

Abstract Background: The most severe form of β-thalassemia, β°-thalassemia major, is characterized by the complete absence of normal β-globin chain, and is often lethal. Autologous transplantation of genetically-modified hematopoietic stem cells (HSCs) using lentiviral vectors have been used successfully to achieve clinical efficacy in one patient, although clonal expansion of a myeloid cell population also occurred in this patient which was associated with the activation of a cellular proto-oncogene, HMGA2. We reasoned that recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus (AAV), might offer a safer alternative. We have previously documented that although the conventional single-stranded (ss) AAV2 vectors mediated β-globin gene transfer and expression in primary human fetal liver cells and in human HSCs from patients with β-thalassemia patients in vitro, the level of transgene expression was sub-optimal. In the present study, we investigated whether double-stranded self-complementary (sc) AAV2 vectors could overcome this limitation. Methods: Human HSCs, obtained from a β-thalassemia homozygous patient, were mock-transduced or transduced with recombinant scAAV2-β-globin vectors at 5×104 vgs/cell, followed by i.v. injection into sub-lethally irradiated NOD/SCID mice (2.65 cGy total body irradiation), which were also pre-treated with 200 µg purified anti-IL2RB/CD122 monoclonal antibody. Recipient mice were sacrificed 12 weeks post-transplantation. Bone marrow cells from recipient mice were analyzed by BFU-E assays. Human β-globin gene expression in human erythroid progenitor cells from transplanted mice was evaluated by RT-PCR. Results: Pre-treatment of NOD/SCID mice with anti-CD122 antibody improves engraftment of human HSCs in bone marrow of receipt mice. Human β-actin (538-bp) and β-globin (272-bp) transcripts were detected by RT-PCR in bone marrow cells from all recipient mice, indicating that recombinant scAAV2-β-globin–transduced HSCs from a patient with β-thalassemia were successfully transduced and transplanted in these mice and that human β-globin gene was transcriptionally active 12 weeks post-transplantation. Conclusion: Our results indicate that human HSCs from β-thalassemia patients can be efficiently transduced by recombinant scAAV2-β-globin vectors followed by expression of normal human β-globin gene. These studies provide the proof-of-concept that scAAV2 vector-mediated gene transfer into human HSCs might be a potentially safer alternative approach for gene therapy of β-thalassemia. Disclosures No relevant conflicts of interest to declare.

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Distinct Effects of SCF and Hes1 On Normal Hematopoietic Stem and Progenitor Cells During Leukemic Cell Expansion in a T-ALL Mouse Model

Blood ◽

10.1182/blood.v120.21.1216.1216 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1216-1216

Author(s):

Chen Tian ◽

Zhipan Cao ◽

Qiao Li ◽

Jinhong Wang ◽

Zhenyu Ju ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Progenitor Cells ◽

Real Time ◽

Hematopoietic Cells ◽

Leukemic Cell ◽

Leukemic Cells ◽

Rt Pcr ◽

Hematopoietic Stem

Abstract Abstract 1216 During leukemia development, emerging leukemic cells out-compete normal hematopoietic cells and become predominant in the body. How hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) respond to the growth of leukemic cell population is an important, yet less investigated area. Our previous study demonstrated differential effects of a leukemic environment on normal HSCs and HPCs in the Notch1-induced T-ALL mouse model (Hu X, et al. Blood 2009). We found that normal HSCs were better preserved in the leukemic bone marrow in part due to increased quiescence of the HSCs and in contrast, HPCs were exhausted during the expansion of leukemic cells. Our current work is aimed to further explore the molecular mechanisms concerning the distinct impacts of leukemic environment on normal HSCs and HPCs in the T-ALL mouse model. Given the previous report by others showing that increased secretion of stem cell factor (SCF) by myeloid leukemia cells played an important role in inducing normal HSCs/HPCs out of their niche and thus allowing leukemic cells to occupy the niche in the human-NOD/SCID xeno-graft model (Sipkins DA et al, Science 2008), we first examined the expression of SCF by ELISA, Western blot and real-time RT PCR in both normal hematopoietic and leukemic cell fractions in the Notch1-induced T-ALL mouse model as previously reported. We found that while expression of SCF in peripheral blood (PB) or bone marrow (BM) was increased in the leukemic mice, both mRNA and protein levels of SCF in normal hematopoietic cells were higher than that in leukemic cells, thereby suggesting that elevated SCF might be mainly secreted by non-leukemic cells in the leukemic hosts of our model. Further assessments on the role of SCF in leukemogenesis with the mice specifically deficient in SCF in different niche cell types are currently under investigation in our laboratory. In order to define potential mediators in HSCs in response to leukemic cell growth, a microarray study on normal HSCs isolated from T-ALL leukemic mice and the control mice was conducted. Gene expression profiling showed significantly differed expression of 169 genes (127 up and 42 down). Especially, real-time RT PCR confirmed an increase of Hes1, p21, Fbxw11, IL-18R1 and Itgb3, and a decrease of CXCR4 and Mmp2. Interestingly, the expression of Hes1 and its target gene, p21 were elevated in normal HSCs but not in HPCs, letting us to hypothesize that Hes1 might be in part mediate the different responses of HSCs and HPCs to the T-ALL leukemic environment. To test this hypothesis, we ectopically expressed Hes1 in normal hematopoietic cells and then examined their functions under the leukemic condition. BM cells from B6.SJL mice were transduced with either MSCV-Hes1-IRES-GFP or control MSCV-GFP vector. After transduction, Hes1-GFP+or control-GFP+cells were co-transplanted with the Notch1-induced T-ALL cells into lethally irradiated C57BL/6J recipients. The engrafted cells from the leukemic BM were analyzed and Hes1-GFP+or control-GFP+cells were sorted for functional assessments. Interestingly, although over-expression of Hes1 inhibited the growth of colony forming cell (CFC) in vitro, it could potentiate the long-term repopulating cells by maintaining more cells in the quiescent (G0) state in vivo. Taken together, our current study supports a role of Hes1 in mediating the distinct responses of normal HSCs and HPCs to the T-ALL leukemic environment. Disclosures: No relevant conflicts of interest to declare.

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Posttransplantation Bone Marrow Assessment by Quantifying Hematopoietic Cell–Derived mRNAs in Plasma Exosomes/Microvesicles

Clinical Chemistry ◽

10.1373/clinchem.2013.213850 ◽

2014 ◽

Vol 60 (4) ◽

pp. 675-682 ◽

Cited By ~ 20

Author(s):

Jun Aoki ◽

Kazuteru Ohashi ◽

Masato Mitsuhashi ◽

Taku Murakami ◽

Melanie Oakes ◽

...

Keyword(s):

Bone Marrow ◽

Human Plasma ◽

Medical Procedure ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Reverse Transcription Pcr ◽

Hematological Analysis ◽

Hematopoietic Precursor ◽

Specific Mrnas ◽

New Biomarkers

Abstract BACKGROUND Bone marrow (BM) aspiration often can be a painful medical procedure. It is unavoidable, however, because hematopoietic precursor cells (HPC) exist only in BM and few escape to peripheral blood (PB). We hypothesized that HPCs might release exosomes and microvesicles (EMV) in BM, and the resulting EMV would penetrate into PB. Such BM-derived EMV might be identified in PB by measuring specific mRNAs produced by HPC. METHODS Human plasma was applied to an EMV-capture filter plate. After centrifugation, captured EMV were lysed on the filter plate. Resulting lysates were transferred to an oligo(dT)-immobilized microplate for mRNA isolation followed by reverse transcription PCR (RT-PCR). Using this system, myeloid-, erythroid-, and megakaryocyte-lineage–specific poly(A)+ mRNAs were quantified in plasma obtained from 18 patients who had undergone hematopoietic stem cell transplantation (HSCT). RESULTS When fluorescent liposomes were applied to the filter plate, more than 95% of applied liposomes were absorbed. When human plasma was applied, a scanning electron microscope showed EMV-like particles on the membrane of the filter plate. After RT-PCR, various HPC-specific mRNAs were detected, and the results were equivalent to those derived from the standard ultracentrifugation method. The levels of these mRNAs were undetectable after HSCT and became detectable 1–2 weeks after HSCT, a substantially earlier time point than with traditional hematological analysis. The recovery of EMV mRNA at day 15 corresponded to the final clinical outcome at day 180. CONCLUSIONS HPC-derived mRNAs in plasma EMV may represent new biomarkers for the assessment of BM condition and could reduce the necessity for frequent BM aspiration.

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Gfi-1 Is Overexpressed on Chronic Myeloid Leukemic Cells.

Blood ◽

10.1182/blood.v110.11.2928.2928 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2928-2928

Author(s):

Hui Cheng ◽

Wei Chang ◽

Ming Huang ◽

Ying Sun ◽

Yicheng Zhang

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Fluorescence Intensity ◽

Zinc Finger Protein ◽

Interleukin 2 ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Control Samples

Abstract Gfi-1 oncogene was first discovered as an integration site for Moloney murine leukemia virus(MoMulv) in virally infected cells that were selected for interleukin-2 (IL-2) independence; In synergy with pim-1 and L-Myc genes, Gfi-1 was involved in the pathogenesis of lymphoid malignancies. Recent studies also indicated that Gfi-1 regulates self-renewal and engraftment of hematopoietic stem cells and the development of neutrophils. Normally, Gfi-1 zinc finger protein is expressed almost exclusively on thymocytes or stem cells. Chronic myeloid leukemia (CML) is a typical hematopoietic stem cell disease, we ask whether Gfi-1 is expressed on CML cells and the possible role in the leukemogenesis. By RT-PCR and flow cytometry, we analysed the expression of Gfi-1 in the transcription and the protein levels respectively on bone marrow mononuclear cells(MNC) from 53 CML patients. Control samples were taken from the ribs of 5 patients undergoing thoracic surgery for lung cancer. Results Gfi-1 mRNA was detected in K562 cell line and all the MNCs from CML patients but not or very weak in control MNCs; Flow cytometry study show that both the positive percentage (87.2±7.5% Vs 12.4±6.6%, p<0.01) and the relative fluorescence intensity (represented by mean fluorescence intensity, 58.94 ±22.09 Vs 18.67±7.88, p<0.01) were significantly increased in CML MNCs. Considering the complexity of the composition of bone marrow MNCs, we also sorted CD34+ cells from CML or control samples and examined by Rt-PCR and flow cytometry; The Gfi-1 was found to be overexpressed in CD34+ cells of CML; On confocal microscopy, Gfi-1 was mainly sublocalized in nucleus. Conclusion Gfi-1 was overexpressed in MNCs or CD34+ cells from CML patients; Gfi-1 may be implicated in the leukemogenesis of CML and potentially be targeted in the future for treatment of CML.

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