Culture of Mobilized Human CD34+ Cells in Hypoxic Conditions Improves Lentiviral Transduction Efficiency in SCID-Repopulating Cells

Abstract Under normal physiological conditions, hematopoietic stem cells (HSC) are sequestered in a hypoxic microenvironment in the bone marrow (BM), suggesting that low oxygen levels may play a fundamental role in the maintenance of normal stem cell function and protect these cells from the damaging effects of reactive oxygen species (ROS). In vitro culture of human BM CD34+ cells under hypoxic conditions has been shown to result in expansion of SCID-repopulating cells (SRC) as compared to culture under normoxic conditions (JCI112 (1); 126, 2003). We investigated whether culture of human mobilized CD34+ cells under low oxygen conditions (5% O2) could improve lentiviral transduction efficiency in SRC compared with culture under atmospheric O2 conditions (21%). G-CSF mobilized CD34+ cells from 4 healthy volunteers were prestimulated for 48 hours in the presence of cytokines (SCF, Flt-3 ligand and thrombopoietin) and subsequently transduced in fibronectin coated plates for 24 hours with SIN-lentiviral vectors carrying the GFP gene under the control of an EF1α promoter. In 3 experiments, cells were used for in vitro assays, including ROS, phenotypic, cell cycle, clonogenic and apoptosis assays. In one experiment, cells were injected intravenously in the tail vein of sublethally irradiated NOD/SCID IL2rγ −/− mice after transduction. Intracellular ROS levels increased more significantly in human CD34+ cells cultured for 3 days in 21% O2 compared with cells cultured in 5% O2. When cultures were maintained more than 3 days, ROS levels were similar between the 2 conditions. The levels of expansion of CD34+ cells compared with baseline were similar in hypoxia (3.9-fold) and normoxia (3.5-fold) (p=0.47). In contrast, the expansion of CD34+CD38− cells, a subpopulation enriched in HSCs, was greater in hypoxia (3.8-fold) than in normoxia (2.2-fold) (p=0.02). After 3 days of culture, the total number of colony-forming cells (CFC) increased 1.1-fold and 1.3-fold under hypoxic and normoxic conditions, respectively (p=0.32) compared with freshly isolated CD34+ cells. The level of O2 had no significant effect on lineage commitment of the CFC. At baseline, the majority (59.5%) of the CD34+ cells were in the G0 phase of the cell cycle. After 3 days in culture under hypoxic or normoxic conditions, the percentages of cells in G0 were 5.5% and 3.5%, respectively (p=0.03). The differences in percentages of cells in the G1 and G2/S/M phases of the cell cycle were not statistically different. The percentages of CD34+ apoptotic cells were similar between hypoxic (32.8%) and normoxic (29.5%) conditions (p=0.18). The pO2 also had no impact on CD34+ cell death (12.2% at 5% O2 and 11.7% at 21% O2, p=0.9). When considering the bulk of CD34+ cells after transduction with GFP-lentiviral vectors, there was no statistically significant difference in the percentages of GFP+ cells under hypoxia (22.3%) or normoxia (21%) (p=0.88). In contrast, when CD34+ cells cultured under hypoxia were injected into NOD/SCID IL2rγ −/− mice at the end of the transduction period, improved human cell engraftment and lentiviral transduction efficiency were detected 2 months after transplantation compared with CD34+ cells cultured under normoxia. Human cell engraftment in the mouse BM, as determined by flow cytometry using a human specific CD45 antibody, was 84% in the hypoxic group (n=4) and 54% in the normoxic group (n=4) (p=0.04). The level of O2 had no significant impact on the lineage commitment of the SRC, with a majority of CD45+CD15+ granulocytes in both groups. The percentage of GFP+CD45+ cells was 54% (hypoxia) and 43% (normoxia) (p=0.02), indicating an improved transduction efficiency of SRC under hypoxic conditions. Overall, these data indicate that human CD34+ cells cultured under low oxygen conditions maintain a more primitive phenotype and have an increased susceptibility to lentiviral transduction compared with cells cultured in 21% O2 conditions. Improved engraftment and transduction efficiency do not appear to be related to decreased apoptosis in lower O2 concentrations; instead, increased ROS production in higher O2 concentrations could lead to increased cell signaling and differentiation. Use of low O2 levels for in vitro transduction of human CD34+ cells could have important clinical implications in gene therapy.

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Culture Of Mobilized Rhesus Macaque CD34+ Cells In Hypoxic Conditions Does Not Improve Lentiviral Transduction Efficiency In Long-Term Repopulating Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v122.21.5557.5557 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5557-5557

Author(s):

Andre Larochelle ◽

Ayla Cash ◽

Zanetta Chang ◽

Brian Ichwan ◽

Jean-Yves Metais ◽

...

Keyword(s):

Cell Cycle ◽

Gene Therapy ◽

Rhesus Macaque ◽

Lentiviral Vectors ◽

Low Oxygen ◽

Lentiviral Transduction ◽

Hypoxic Conditions ◽

Cd34 Cells

Abstract Low oxygen levels have been proposed to play a fundamental role in the maintenance of normal hematopoietic stem cells (HSC) function. We investigated whether culture of rhesus macaque mobilized CD34+ cells under low oxygen conditions (1-5% O2) could improve lentiviral transduction efficiency in HSCs compared to culture under atmospheric O2 conditions (21%). Mobilized CD34+ cells from 4 animals were prestimulated for 12 hours in the presence of cytokines and subsequently divided in two equal fractions for transduction with SIN-lentiviral vectors (MOI=100) expressing GFP or TdTomato fluorescent proteins for 48 hours. In 3 animals, cells were transduced with GFP vectors in hypoxia and TdTomato vectors in normoxia. These conditions were reversed in one animal. A portion of the transduced cells was used for phenotypic, cell cycle, clonogenic and apoptosis assays. The remaining cells from both fractions were combined and injected intravenously in lethally irradiated rhesus macaques and in vivo gene marking was measured in peripheral blood granulocytes for up to 21 months after transplantation. The numbers of total live cells and CD34+ cells after 2.5 days in culture were not significantly different compared to baseline for both hypoxic and normoxic conditions (p=0.85 and p=0.84, respectively). Similarly, numbers of CD34+CD38- cells were stable compared to baseline in hypoxia and normoxia (p=0.94). After 2.5 days of culture, the total number of colony-forming cells increased 1.4-fold under both hypoxic and normoxic conditions (p=0.69) compared to freshly isolated CD34+ cells. At baseline, the majority (50.6 + 4.4%) of the CD34+ cells were in the G0 phase of the cell cycle. After 2.5 days in culture under hypoxic or normoxic conditions, the percentages of cells in G0 were 19.9 + 8.8% and 18.6 + 5.9%, respectively (p=0.82). The differences in percentages of cells in the G1 and S/G2/M phases of the cell cycle were also not statistically significant (p=0.175 and p=0.732, respectively). The pO2 had no impact on cell death (18.2 + 5.3% in hypoxia and 16.8 + 4.8% in normoxia, p=0.69) or on the percentages of apoptotic cells (11.1 + 6.7% in hypoxia and 8.4 + 2.3% in normoxia, p=0.47). When considering the bulk of CD34+ cells after transduction with lentiviral vectors, the percentages of GFP-marked cells were consistently higher (range 1.8 to 2.2-fold, mean 2.1-fold) compared to TdTomato-marked cells independently of the transduction conditions used, consistent with a 2.1-fold intrinsic superiority of GFP-based lentiviral vectors compared to TdTomato vectors at equivalent MOI. Taking into account the inherent vector differences, transduction efficiencies were similar in normoxia (70.7 + 11.7%) and hypoxia (68.0 + 16.3%) (p=0.80). When adjusted for the intrinsic superiority of GFP-based vectors, the contribution to long-term in vivo gene marking measured by flow cytometry and quantitative PCR was comparable between cells transduced in 21% pO2 (9.0 + 4.5%) and 5% pO2 conditions (8.4 + 4.9%) (p=0.87). Consistent with these data, GFP and TdTomato-labeled hematopoietic cells were equally visualized, using a confocal/2-photon hybrid microscopy approach, in BM biopsy specimens collected at various time points after transplantation. Given recent evidence suggesting that lower oxygen concentrations (<1.5%) may be required to stabilize HIF-1a, a key sensor of hypoxic conditions, transduction was performed under extreme hypoxic conditions (1% O2) in one animal. While the overall viability of the bulk CD34+ cells after 2.5 days of transduction under extreme hypoxia was comparable to cells cultured in normoxia, their contribution to long-term in vivo marking was negligible (0.3%) compared to cells transduced under normoxic conditions (2.4%), suggesting toxicity of very low oxygen levels on HSCs. Overall, when current methodologies used for the genetic manipulation of HSCs for gene therapy applications were performed under hypoxic conditions, susceptibility to lentiviral transduction of CD34+ cells was not ameliorated compared to cells cultured in 21% O2 conditions in the rhesus macaque transplantation model. The short culture times (2-3 days) used in recent lentivirus-based gene therapy clinical trials and replicated in this study may not be sufficient to impact the phenotype of long-term repopulating HSCs and do not warrant incorporation of hypoxia in current gene therapy protocols. Disclosures: No relevant conflicts of interest to declare.

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Determining Limitations in Human CD34+ Cell Transduction with An HIV1-Based Lentiviral Vector,

Blood ◽

10.1182/blood.v118.21.4171.4171 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4171-4171 ◽

Cited By ~ 2

Author(s):

Rashidah M. Green ◽

John F. Tisdale ◽

Naoya Uchida

Keyword(s):

Hela Cells ◽

Reverse Transcription ◽

Nuclear Dna ◽

Nuclear Transport ◽

Transduction Efficiency ◽

Lentiviral Transduction ◽

Human Cd34 ◽

Limiting Step ◽

Cd34 Cells ◽

Vector Dna

Abstract Abstract 4171 Hematopoietic stem cell (HSC)-targeted gene therapy approaches utilizing HIV1-based lentiviral vectors is a promising modality for a number of inherited and acquired disorders affecting the blood. Unlike cell lines and murine HSCs, human HSCs demonstrate lower transduction efficiency with HIV1 vectors, potentially limiting this approach. Lentiviral transduction requires four essential steps: (1) internalization, (2) reverse transcription, (3) nuclear transport, and (4) integration into genomic DNA. In this study, we sought to evaluate which step limits transduction of human CD34+ cells with HIV1 vectors. We transduced HeLa cells and human CD34+ cells with a self-inactivating-HIV1 vector at low and a 10-fold-higher multiplicity of infection (MOI) (MOI 0.5 vs. MOI 5 for HeLa cells and MOI 5 vs. MOI 50 in CD34+ cells, respectively) and assayed each of the four steps based upon the rationale that if a given step was not rate limiting, a 10-fold-greater value would be obtained at the 10-fold-higher MOI. Amounts of the vectors were determined by real time PCR using RNA and DNA samples extracted from the transduced cells over time. The ratios of vector genome amounts at high MOI to amounts at low MOI were compared between CD34+ cells and HeLa cells to determine whether there was a limiting step for CD34+ cell transduction. To evaluate RNA internalization, we determined relative amounts of vector RNA in the cells at 5 min, 10 min, 30 min, and 60 min after transduction. Among HeLa cells, relative amounts of vector RNA showed a rapid increase and then plateaued at 30 min after transduction, while in the CD34+ cells, relative amounts of vector RNA increased gradually over 60 min. When we calculated the ratios of vector RNA amounts at high MOI to low MOI for each type of cell, we observed similar ratios when comparing HeLa to CD34+ cells at 60 min after transduction. These data suggest that despite slower internalization of vector RNA in CD34+ cells, it is not a limiting step in lentiviral transduction. To evaluate reverse transcription, nuclear transport, and integration; we determined relative amounts of vector DNA at 2 hrs, 4 hrs, 6 hrs, 12 hrs, 24 hrs, 2 days, 3 days, 6 days, and 10 days after transduction. We detected similar patterns of relative vector DNA in both Hela and CD34+ cells: as their relative amounts increased, they reached a peak at 24 hrs (reverse transcription), decreased, and then reached a plateau at 2–3 days after transduction (nuclear transport and integration). CD34+ cells showed higher ratios of vector DNA at high MOI to low MOI at 2–24 hrs, similar ratios at 2 days, and lower ratios at 3–10 days, compared to HeLa cells (Figure). These data suggest that transduction of human CD34+ cells has a limitation around 2 days after vector exposure corresponding to nuclear transport or integration. To evaluate nuclear transport for both types of cells, we compared the relative amounts of vector DNA to total DNA and nuclear DNA. Nuclear DNA was extracted from the nuclear fractions of transduced cells which had been isolated by a sucrose gradient. In both cells, the nuclear DNA had tendencies to show lower amounts of vector DNA at 6–48 hrs after transduction and similar or higher amounts of vector DNA at 2–7 days, compared to the total DNA. These data suggest that vector DNA is transported to the nucleus in about 2–3 days. Taken together, these data suggest that nuclear transport is a limiting step in lentiviral transduction of human CD34+ cells. We hypothesized that transduction efficiency for human CD34+ cells might increase under conditions that induce greater cell expansion, since dividing cells do not have intact nuclear membranes. We compared relative amounts of vector DNA in human CD34+ cells transduced in X-VIVO10 medium (standard) and StemlineII medium (greater expansion) at 12 hrs, 24 hrs, 2 days, 3 days, 5 days, and 7 days after transduction. StemlineII exposed cells showed higher amounts of vector DNA, compared to X-VIVO10, at all time points except 24 hrs. The data suggest that StemlineII increases transduction efficiency for human CD34+ cells by increasing nuclear transport. In summary, we demonstrate that nuclear transport is a limiting step in lentiviral transduction of human CD34+ cells and that StemlineII medium could increase transduction efficiency. These data are helpful for the design of strategies to improve upon lentiviral transduction for human CD34+ cells by improving nuclear transport. Disclosures: No relevant conflicts of interest to declare.

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Convergence Of Hypoxia and TGFβ Pathways On Cell Cycle Regulation In Human Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood.v122.21.3694.3694 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3694-3694

Author(s):

Albertus T.J. Wierenga ◽

Edo Vellenga ◽

Jan Jacob Schuringa

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

G Protein ◽

Gene Transcription ◽

Target Genes ◽

Low Oxygen ◽

Hematopoietic Stem ◽

Gm Csf ◽

Cd34 Cells ◽

Oxygen Conditions

Abstract Hematopoietic stem cells reside within specialized hypoxic niches in the bone marrow where they are kept in a relative quiescent state. One of the key pathways activated under low oxygen conditions is the Hypoxia Induced Factor (HIF) pathway. HIF1 and HIF2 act as oxygen sensors that are degraded by the Von Hippel Lindau (VHL) tumor suppressor protein under normoxic conditions but not when oxygen levels are low, resulting in stabilization of the HIF proteins, translocation to the nucleus and initiation of target gene transcription. Although it has been shown that HIF1 and 2 fulfill essential roles in the regulation of HSC fate, little is known about the mechanisms that are involved. Here, we set out to investigate the effects of hypoxia, HIF1 and HIF2 on gene transcription in human hematopoietic stem/progenitor cells. Cord blood (CB) CD34+ cells were cultured under low oxygen conditions (2%), or were lentivirally transduced with constitutively active HIF1(P402/564) and HIF2(P405/531) constructs under normoxic conditions and after 24 hrs transcriptome changes were analyzed by Illumina BeadArray analysis. This provided the possibility to identify common hypoxia-HIF1-HIF2 gene signatures, but also the identification of specific target genes that were exclusively regulated by HIF1, HIF2 or hypoxia. Geneset enrichment analysis (GSEA) using Gene Ontology genesets revealed that overexpression of HIF1 and -2 resulted in significant enrichment for known pathways like “hypoxia induced signaling”, but unexpectedly also for the Transforming Growth Factor beta (TGFβ) pathway. GSEA using a published dataset of TGFβ stimulated CB CD34+ cells indeed confirmed a high correlation between hypoxia target genes and TGFβ induced genes. Two of the most significantly upregulated genes in both gene sets were the cyclin dependent kinase inhibitor CDKN1C (p57kip2) and Regulator of G-protein signaling (RGS)1. q-RT-PCR analysis demonstrated enhanced expression of CDKN1C by hypoxia treatment or HIF overexpression under normoxia in combination with TGFβ stimulation. Although it was demonstrated that CD34+cells cultured under hypoxic conditions secreted high levels of latent TGFβ, no rescue of the hypoxia induced cell cycle arrest was demonstrated by knockdown of SMAD4, arguing against direct effects of hypoxia-induced secreted TGFβ on cell cycle quiescence. RGS1 is a member of the RGS family, involved in the negative regulation of G-protein coupled receptor signaling. Overexpression studies under normoxic conditions in CB CD34+cells demonstrated a decrease of SDF1-mediated migration. Furthermore, overexpression of RGS1 attenuated SDF1 and GM-CSF-induced ERK phosphorylation whereas the GM-CSF-induced STAT5 tyrosine phosphorylation was unaffected. These findings indicate that RGS1 can interfere with specific signaling pathways involved in the regulation of cell proliferation and migration. Analysis of the CDKN1C as well as the RGS1 promoters revealed binding sites for both HIF and SMAD2/3/4 in the proximal part, suggesting that both pathways can indeed converge on the regulation of these important proteins that control cell cycle progression and the response to stimulatory cytokines in human stem/progenitor cells. Disclosures: No relevant conflicts of interest to declare.

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Functional Heterogeneity of Human CD34+ Cells Isolated in Subcompartments of the G0 /G1 Phase of the Cell Cycle

Blood ◽

10.1182/blood.v90.11.4384.4384_4384_4393 ◽

1997 ◽

Vol 90 (11) ◽

pp. 4384-4393 ◽

Cited By ~ 6

Author(s):

André Gothot ◽

Robert Pyatt ◽

Jon McMahel ◽

Susan Rice ◽

Edward F. Srour

Keyword(s):

Cell Cycle ◽

Ex Vivo ◽

G1 Phase ◽

Dna And Rna ◽

Human Cd34 ◽

Rna Content ◽

Cd34 Cells ◽

Ex Vivo Culture

Using simultaneous Hoechst 33342 (Hst) and Pyronin Y (PY) staining for determination of DNA and RNA content, respectively, human CD34+ cells were isolated in subcompartments of the G0 /G1 phase of the cell cycle by flow cytometric cell sorting. In both bone marrow (BM) and mobilized peripheral blood (MPB) CD34+ cells, primitive long-term hematopoietic culture-initiating cell (LTHC-IC) activity was higher in CD34+ cells isolated in G0 (G0CD34+ cells) than in those residing in G1 (G1CD34+ cells). However, as MPB CD34+ cells displayed a more homogeneous cell-cycle status within the G0 /G1 phase and a relative absence of cells in late G1 , DNA/RNA fractionation was less effective in segregating LTHC-IC in MPB than in BM. BM CD34+ cells belonging to four subcompartments of increasing RNA content within the G0 /G1 phase were evaluated in functional assays. The persistence of CD34 expression in suspension culture was inversely correlated with the initial RNA content of test cells. Multipotential progenitors were present in G0 or early G1 subcompartments, while lineage-restricted granulomonocytic progenitors were more abundant in late G1 . In vitro hematopoiesis was maintained for up to 6 weeks with G0CD34+ cells, whereas production of clonogenic progenitors was more limited in cultures initiated with G1CD34+ cells. To test the hypothesis that primitive LTHC-ICs would reenter a state of relative quiescence after in vitro division, BM CD34+ cells proliferating in ex vivo cultures were identified from their quiescent counterparts by a relative loss of membrane intercalating dye PKH2, and were further fractionated with Hst and PY. The same functional hierarchy was documented within the PKH2dim population whereby LTHC-IC frequency was higher for CD34+ cells reselected in G0 after in vitro division than for CD34+ cells reisolated in G1 or in S/G2 + M. However, the highest LTHC-IC frequency was found in quiescent PKH2bright CD34+ cells. Together, these results support the concept that cells with distinct hematopoietic capabilities follow different pathways during the G0 /G1 phase of the cell cycle both in vivo and during ex vivo culture.

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TRIM5α Variation Affects Lentiviral Transduction Efficiency for Long-Term Hematopoietic Repopulating Cells in a Rhesus Stem Cell Transplantation Model

Blood ◽

10.1182/blood.v118.21.2056.2056 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2056-2056

Author(s):

Naoya Uchida ◽

Matthew M. Hsieh ◽

Aylin C. Bonifacino ◽

Sandra D. Price ◽

Allen E. Krouse ◽

...

Keyword(s):

Stem Cell ◽

Cell Lines ◽

Transduction Efficiency ◽

Gfp Expression ◽

Lentiviral Transduction ◽

Cd34 Cells ◽

Hiv 1

Abstract Abstract 2056 The Tripartite Motif-containing protein 5 α (TRIM5α) is thought to play an important role in restricting retroviral infection among species in nature, and restricts HIV-1 based lentiviral transduction of rhesus hematopoietic repopulating cells. We previously developed a chimeric HIV-1 based lentiviral vectors (χHIV vectors) which includes a simian immunodeficiency virus (SIV) capsid in place of the HIV-1 capsid to circumvent this restriction (J Virol. 2009). Transduction efficiency, however, remained highly variable between animals. In this study, we sought to evaluate the effects of rhesus TRIM5α polymorphisms on lentiviral transduction of rhesus hematopoietic repopulating cells. To evaluate whether rhesus TRIM5α polymorphisms influence the efficiency of lentiviral transduction, we transduced cell lines expressing 6 different rhesus TRIM5α types (Mamu-1, -2, -3, -4, -5, and TrimCyp) with eGFP-encoding HIV1, χHIV, and SIV vectors (Figure). Among all TRIM5α cell lines, transgene expression rates (%GFP) from the χHIV vector fell between that of the HIV-1 vector and that of the SIV vector. For the χHIV and SIV vectors, transduction efficiency was reduced in Mamu-1, -2, and -3 (p<0.01) expressing cell lines when compared to that of control cells. For the HIV-1 vector, there was a reduction in %GFP among all TRIM5α types (p<0.01). These results suggest that both the χHIV and SIV vectors escape restriction through rhesus TRIM5α Mamu-4, -5, and TrimCyp. We then analyzed 16 rhesus macaques who were transplanted with CD34+ cells transduced with the χHIV vector. We evaluated %GFP in granulocytes and lymphocytes 6 months after transplantation, as %GFP expression has been found to plateau in the peripheral blood at 6 months. Transduction efficiency of rhesus CD34+ cells was also evaluated in vitro at the time of transplant based on %GFP expression. For statistical analysis, we assessed factors that potentially affect transduction efficiency, including age, sex, weight, total number of mobilized CD34+ cells, cytokine mobilization regimen (G-CSF & stem cell factor (SCF) vs. G-CSF & plerixafor), cell density during transduction, and TRIM5α polymorphisms. Multivariable analysis demonstrated that TRIM5α type Mamu-4 (50.9±19.0% vs. 26.6±16.7%, p=0.04) as well as mobilization regimen (48.5±17.4% vs. 12.7±7.1%, p=0.01) affected CD34+ cell transduction efficiency in vitro. TRIM5α Mamu-4 only showed significant effects on %GFP among lymphocytes in vivo (23.7±17.9% vs. 5.3±3.1%, p=0.046). When analyzing the %GFP among granulocytes in vivo, there was a significant correlation with weight (p=0.01), mobilized CD34+ cell number (p=0.02), TRIM5α type Mamu-4 (29.3±25.4% vs. 8.4±6.0%, p=0.03), and mobilization regimen (25.4±24.1% vs. 7.5±3.0%, p=0.04). If in vitro %GFP is included in the analysis, both %GFP among granulocytes and lymphocytes are strongly affected (<0.001) and TRIM5α type Mamu-4 and mobilization regimen are no longer significant. Univariate analysis showed similar tendencies regarding %GFP among granulocytes and lymphocytes. Taken together, our data suggest that TRIM5α type Mamu-4, mobilization regimen (G-CSF & SCF), and CD34+ cell transduction efficiency in vitro are important factors that can predict higher %GFP in granulocytes and lymphocytes 6 months following transplantation. In summary, rhesus TRIM5α polymorphisms (especially type Mamu-4) play an important role in allowing efficient lentiviral transduction of long-term repopulating cells and contribute to the variable results observed in the rhesus hematopoietic stem cell gene therapy model. Disclosures: No relevant conflicts of interest to declare.

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Self-Renewal and Differentiation in Hematopoietic Stem and Progenitor Cells Is Controlled By the APC/C Coactivator Cdh1

Blood ◽

10.1182/blood.v126.23.2370.2370 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2370-2370

Author(s):

Daniel Ewerth ◽

Stefanie Kreutmair ◽

Birgit Kügelgen ◽

Dagmar Wider ◽

Julia Felthaus ◽

...

Keyword(s):

Cell Cycle ◽

Research Funding ◽

Self Renewal ◽

Hematopoietic Stem ◽

Nsg Mice ◽

Human Cd34 ◽

Cd34 Cells ◽

Stem And Progenitor Cells

Abstract Introduction: Hematopoietic stem and progenitor cells (HSPCs) represent the lifelong source of all blood cells and continuously renew the hematopoietic system by differentiation into mature blood cells. The process of differentiation is predominantly initiated in G1 phase of the cell cycle when stem cells leave their quiescent state. During G1 the anaphase-promoting complex or cyclosome (APC/C) associated with the coactivator Cdh1 is highly active and marks proteins for proteasomal degradation to regulate proliferation. In addition, Cdh1 has been shown to control terminal differentiation in neurons, muscle cells or osteoblasts. Here we show that Cdh1 is also a critical regulator of human HSPC differentiation and self-renewal. Methods: Human CD34+ cells were collected from peripheral blood (PB) of G-CSF mobilized donors and cultured in the presence of different cytokine combinations. To analyze cell division and self-renewal versus differentiation, CFSE staining was used in combination with flow cytometric detection of CD34 expression. The knockdown and overexpression of Cdh1 was achieved by lentiviral delivery of suitable vectors into target cells. After cell sorting transduced (GFP+) CD34+ cells were used for in vitro differentiation in liquid culture or CFU assay. For in vivo experiments purified cells were transplanted into NSG mice. Results: G-CSF mobilized CD34+ cells showed effective differentiation into granulocytes (SCF, G-CSF), erythrocytes (SCF, EPO) or extended self-renewal (SCF, TPO, Flt3-L) when stimulated in vitro. The differentiation was characterized by a fast downregulation of Cdh1 on protein level, while Cdh1 remained expressed under self-renewal conditions. A detailed analysis of different subsets, both in vitro and in vivo, showed high Cdh1 level in CD34+ cells and low expression in myeloid cells. Analysis of proliferation revealed lowest division rates during self-renewal, accompanied by higher frequency of CD34+ cells. The fastest proliferation was found after induction of erythropoiesis. These experiments also showed a more rapid decrease of HSPCs' colony-forming ability and of CD34+ cells during granulopoiesis after 2-3 cell divisions in contrast to a moderate decline under self-renewal conditions. The depletion of Cdh1 (Cdh1-kd) had no effect on total cell numbers or proliferation detected by CFSE during differentiation and self-renewal, but showed an increase in S phase cells. These results were confirmed at the single cell level by measuring the cell cycle length of individual cells. Independent of cell cycle regulation, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with lower frequency of Glycophorin A+ cells. In CFU assays, the Cdh1-kd resulted in less primary colony formation, notably CFU-GM and BFU-E, but significantly more secondary colonies compared to control cells. These results suggest that the majority of cells reside in a more undifferentiated state due to Cdh1-kd. The overexpression of Cdh1 showed reversed results with less S phase cells and tendency to increased differentiation in liquid culture and CFU assays. To further validate our results in vivo, we have established a NSG xenotransplant mouse model. Human CD34+ cells depleted of Cdh1 engrafted to a much higher degree in the murine BM 8 and 12 weeks after injection as shown by higher frequencies of human CD45+ cells. Moreover, we also found an increased frequency of human CD19+ B cells after transplantation of CD34+ Cdh1-kd cells. These results suggest an enhanced in vivo repopulation capacity of human CD34+ HSCs in NSG mice when Cdh1 is depleted. Preliminary data in murine hematopoiesis support our hypothesis showing enhanced PB chimerism upon Cdh1-kd. Looking for a mediator of these effects, we found the Cdh1 target protein TRRAP, a cofactor of many HAT complexes, increased upon Cdh1-kd under self-renewal conditions. We use currently RT-qPCR to determine, if this is caused by a transcriptional or post-translational mechanism. Conclusions: Loss of the APC/C coactivator Cdh1 supports self-renewal of CD34+ cells, represses erythropoiesis in vitro and facilitates engraftment capacity and B cell development of human HSPCs in vivo. This work was supported by Josè Carreras Leukemia Foundation grant DCJLS R10/14 (to ME+RW) Disclosures Ewerth: Josè Carreras Leukemia Foundation: Research Funding. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

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The Role of Cyclin C on Hematopoietic Cells.

Blood ◽

10.1182/blood.v108.11.4238.4238 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4238-4238

Author(s):

Yasuhiko Miyata ◽

Yan Lie ◽

Goro Sashida ◽

Stephen Nimer

Keyword(s):

Cell Cycle ◽

Biological Effects ◽

Human Fibroblasts ◽

Hematopoietic Cells ◽

Transduction Efficiency ◽

Quiescent State ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells ◽

Cyclin C

Abstract The regulation of pluripotent hematopoietic stem cells (HSCs) is essential for the maintenance of multilineage blood production throughout life. The ability to self-renew or differentiate is a critical factor in the regulation of HSC numbers and cell fate, but so is the ability to enter and reside in a quiescent state (G0 phase of the cell cycle). The regulation of the G0 stem cell compartment is important for maintaining an inexhaustible pool of HSCs, protected from cellular stress. We have recently shown that the ETS transcription factor MEF is an important regulator of HSC quiescence (H. D. Lacorazza, Cancer Cell, 2006). The mechanism by which MEF or cell cycle regulatory proteins like p21 control entry into (and exit from) the G0 stage of the cell cycle is poorly understood. Insights into the mechanisms of HSC regulation could have important therapeutic potential. Recently, a Cyclin C/cdk3 interaction was reported to play an important role in cell cycle re-entry from quiescent state (G0 to G1) by phosphorylating the retinoblastoma protein (Rb) in human fibroblasts (S. Ren, Cell, 2004). To elucidate if cyclin C is involved in the regulation of cell cycle in hematopoietic cells, especially human CD34+ HSCs, we have optimized a FG12 lentivirus based shRNA strategy (kindly provided by D. Baltimore, PNAS, 2002). shRNA directed against cyclin C successfully decreased both cyclin C mRNA and protein expression levels. Using this vector, we have consistently achieved a 90 % transduction efficiency and a 60–70 % reduction of cyclin C levels in human CD34+ cells as evidenced by real time PCR. Now we are analyzing the cell cycle and biological effects of cyclin C knockdown and overexpression on hematopoietic cells using both human CD34+ cells and human leukemic cell lines.

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Functional Heterogeneity of Human CD34+ Cells Isolated in Subcompartments of the G0 /G1 Phase of the Cell Cycle

Blood ◽

10.1182/blood.v90.11.4384 ◽

1997 ◽

Vol 90 (11) ◽

pp. 4384-4393 ◽

Cited By ~ 148

Author(s):

André Gothot ◽

Robert Pyatt ◽

Jon McMahel ◽

Susan Rice ◽

Edward F. Srour

Keyword(s):

Cell Cycle ◽

Ex Vivo ◽

G1 Phase ◽

Dna And Rna ◽

Human Cd34 ◽

Rna Content ◽

Cd34 Cells ◽

Ex Vivo Culture

Abstract Using simultaneous Hoechst 33342 (Hst) and Pyronin Y (PY) staining for determination of DNA and RNA content, respectively, human CD34+ cells were isolated in subcompartments of the G0 /G1 phase of the cell cycle by flow cytometric cell sorting. In both bone marrow (BM) and mobilized peripheral blood (MPB) CD34+ cells, primitive long-term hematopoietic culture-initiating cell (LTHC-IC) activity was higher in CD34+ cells isolated in G0 (G0CD34+ cells) than in those residing in G1 (G1CD34+ cells). However, as MPB CD34+ cells displayed a more homogeneous cell-cycle status within the G0 /G1 phase and a relative absence of cells in late G1 , DNA/RNA fractionation was less effective in segregating LTHC-IC in MPB than in BM. BM CD34+ cells belonging to four subcompartments of increasing RNA content within the G0 /G1 phase were evaluated in functional assays. The persistence of CD34 expression in suspension culture was inversely correlated with the initial RNA content of test cells. Multipotential progenitors were present in G0 or early G1 subcompartments, while lineage-restricted granulomonocytic progenitors were more abundant in late G1 . In vitro hematopoiesis was maintained for up to 6 weeks with G0CD34+ cells, whereas production of clonogenic progenitors was more limited in cultures initiated with G1CD34+ cells. To test the hypothesis that primitive LTHC-ICs would reenter a state of relative quiescence after in vitro division, BM CD34+ cells proliferating in ex vivo cultures were identified from their quiescent counterparts by a relative loss of membrane intercalating dye PKH2, and were further fractionated with Hst and PY. The same functional hierarchy was documented within the PKH2dim population whereby LTHC-IC frequency was higher for CD34+ cells reselected in G0 after in vitro division than for CD34+ cells reisolated in G1 or in S/G2 + M. However, the highest LTHC-IC frequency was found in quiescent PKH2bright CD34+ cells. Together, these results support the concept that cells with distinct hematopoietic capabilities follow different pathways during the G0 /G1 phase of the cell cycle both in vivo and during ex vivo culture.

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Cyclin C Regulates the Quiescence of Human CD34+CD38- Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v110.11.1273.1273 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1273-1273

Author(s):

Yasuhiko Miyata ◽

Yan Liu ◽

Vladimir Jankovic ◽

Goro Sashida ◽

Silvia Menendez ◽

...

Keyword(s):

Cell Cycle ◽

Fold Increase ◽

Human Cord Blood ◽

Self Renewal ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells ◽

Cyclin C

Abstract The relative quiescence of adult hematopoietic stem cells (HSCs) at steady state represents an important regulatory mechanism for maintaining their self-renewal and engraftment capacity, as well as their resistance to cytotoxic insults. However, the specific mechanisms regulating the intermittent entry of HSCs into the cell cycle are not well characterized. Here we provide the evidence that cyclin C (CCNC) specifically promotes the G0/G1 transition of human CD34+CD38- HSCs, and thus can significantly affect the loss of HSC self-renewal capacity in in vitro culture. Based on the recently hypothesized specific function of CCNC in G0 exit of human fibroblasts, we have analyzed the effects of CCNC loss on the behavior of human cord blood HSCs. We achieved a highly efficient knockdown of CCNC expression (>90%) using lentiviral shRNA (shCCNC) transduction of freshly isolated human cord blood CD34+ cells, allowing the in vitro assessment of early cell cycle regulation in HSCs. First, we observed a 3-fold increase in the G0 fraction of shCCNC transduced CD34+ cells compared to the empty vector control, based on the Pyronin Y and Hoechst 33342 staining 72h after infection. The depletion of CCNC did not prevent cell cycle progression beyond the G1 entry, as we observed no significant changes in the G1/S/G2-M distribution, indicating that critical CCNC activities may be restricted to the G0 checkpoint. Consistent with the reported enrichment of functional HSCs in the G0 fraction, CCNC knockdown (CCNC KD) cells showed increased activity in all surrogate in vitro assays of stem cell-ness tested: a ∼3 fold increase in CD34+ population after long term culture, a ∼2.5 fold increase in long-term culture initiating cells (LTC-ICs) and a ∼3.5 fold increase in cobblestone area forming cells (CAFCs). In contrast, CFU assays using freshly sorted shCCNC cells (and cells obtained after one-week culture in cytokines) showed only a minimal decrease in total colony number, with no difference in colony composition or morphology, indicating no significant effect on hematopoietic progenitor cell differentiation. However, we did observe a prominent effect on secondary CFUs after 2 and 3 weeks in liquid culture (i.e. using the delta assay), namely a 2-fold and 30-fold increase in shCCNC over control culture respectively, again indicating a specific function of CCNC on the more primitive cells. Consistently, CCNC KD robustly enhanced CD34 expression and secondary CFU maintenance in sorted CD34+CD38- cells (HSCs); both markers of hematopoietic cell immaturity were rapidly lost in CD34+CD38+ cells (i.e. the committed progenitor cells) with no detectable effect of shCCNC transduction. Finally, we have found that these effects of CCNC depletion are likely the result of its initial loss of function, as transient CCNC KD, using siRNA transfection of CD34+ cells, produced similar biological effects as the constitutive lentiviral shCCNC expression. Collectively, these data indicate a cell context-dependent effect of CCNC KD on the initial rate of cell cycle entry by quiescent HSCs and suggest that this approach could be used to preserve their functional capacity in culture, potentially enhancing the ex vivo expansion of HSCs, as well as their use in gene therapy protocols. Transplantation of transduced CD34+ cells into sublethally irradiated immunodeficient mice is now under way to establish the potentially beneficial effects of CCNC KD on the engraftment and repopulating capability of cultured HSCs.

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Differential Maintenance of Cell Cycle Status of Human Bone Marrow- and Umbilical Cord Blood-Derived CD34+ Cells after Homing to the Bone Marrow Microenvironment.

Blood ◽

10.1182/blood.v112.11.1327.1327 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1327-1327

Author(s):

Bradley A. Poteat ◽

Brahmananda Reddy Chitteti ◽

Karen E. Pollok ◽

Attaya Suvannasankha ◽

Edward F. Srour

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Cord Blood ◽

Umbilical Cord Blood ◽

Cell Cycle Progression ◽

Cycle Progression ◽

Cd34 Cells ◽

Cell Cycle Status ◽

Cells Cultured

Abstract Transplanted human bone marrow (BM) CD34+ cells remain mitotically quiescent up to 72h following their homing to the BM of irradiated NOD/SCID mice (Blood2002;99(5):1585). To investigate whether umbilical cord blood (UCB)-derived CD34+ cells behave similarly and to assess the impact of the BM microenvironment on this observed temporary cell cycle arrest, we examined the cell cycle status of BM- and UCB-derived CD34+ cells recovered from the BM of irradiated (325 cGy) and non-irradiated NOD/SCID IL2Rγnull (NS2) mice 20h after transplantation (AT). To understand the molecular control of this sustained, but short-lived induced quiescence, expression of cell cycle-related proteins among BM-homed cells was examined by real-time quantitative PCR. Freshly isolated CD34+ cells were stained with CFSE-1 and transplanted into normal or conditioned NS2 mice or cultured in vitro with and without SCF, Flt3, GM-CSF, and IL-3. BM was harvested from NS2 recipients 20h AT and CFSE+ cells were recovered by cell sorting. Cell cycle status was assessed by PI staining in a fraction of recovered CFSE+ and cells cultured in vitro and mRNA was isolated from the remaining cells in both groups. The proportion of cells in G0/G1 phase of cell cycle among BM CD34+ cells was maintained in recipient BM 20h AT regardless of whether the microenvironment was irradiated or not (fresh: 83.7% ± 4.4%, n = 14; irradiated: 82.7% ± 5.3%, n = 6; non-irradiated: 84.4% ± 4.1%, n = 10), suggesting that exit of BM-homed cells from G0/G1 is either a strictly cell intrinsic property or is a phenomenon mediated by microenvironmental modulators present in both intact and injured BM. However, cultured BM CD34+ cells cycled efficiently such that after 20h, only 75.5% ± 5.7%, n = 12 remained in G0/G1. Surprisingly, BM-homed UCB CD34+ cells did not remain quiescent and at 20h AT, only 84.85% ± 13.1% (n = 5) were in G0/G1 compared to 97.0% ± 1.8% (n = 14) for freshly isolated cells, suggesting that different cell cycle regulatory mechanisms control BM versus UCB CD34+ cells. UCB CD34+ cells recovered from the BM of irradiated recipient mice 20h AT had increased levels of Bcl-2 and CDKN1B (p27) while those of CDKN1C (p57) and p53 were decreased relative to those isolated from non-irradiated recipients. These findings suggest that at least in the case of UCB CD34+ cells, different cell cycle regulatory networks may impact cell cycle progression of these cells in an irradiated versus intact BM microenvironment. Compared to freshly isolated BM CD34+ cells, those recovered from the marrow of non-irradiated recipients 20h AT had significantly elevated levels of mRNA for CDKN1a (p21), CDKN1B (p27), p53, and N-cadherin, while the mRNA levels for these molecules in comparable cells cultured in vitro for 20h was relatively unchanged. Taken together, these data illustrate that human CD34+ cells from different tissues behave differently during the first few hours following their homing to the BM and that this behavior may be partially regulated by the status of the microenvironment. Furthermore, data from BM-derived CD34+ cells suggest that following homing, active repression of cell cycle progression may be mediated by induced upregulation of p21 and p27. Mechanisms leading to the upregulation of these cell cycle regulatory molecules remain to be investigated.

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