Generation of Dyskeratosis Congenita-like Hematopoietic Stem Cells through the Stable Inhibition of DKC1

Abstract Dyskeratosis congenita (DC) is a rare telomere biology disorder, which results in different clinical manifestations, including severe bone marrow failure. To date, the only curative treatment for bone marrow failure in DC patients is allogeneic hematopoietic stem cell transplantation. However due to the toxicity associated to this treatment, improved therapies are recommended for DC patients. Here we aimed at generating DC-like human hematopoietic stem cells in which the efficacy of innovative therapies could be investigated. Because X-linked DC is the most frequent form of the disease and is associated with an impaired expression of DKC1, we have generated DC-like hematopoietic stem cells based on the stable knock-down of DKC1 in human CD34 + cells with lentiviral vectors encoding for DKC1 short hairpin RNAs. At a molecular level, DKC1 -interfered CD34 + cells showed a decreased expression of TERC, as well as a diminished telomerase activity and increased DNA damage, cell senescence and apoptosis. Moreover, DKC1 -interfered human CD34 + cells showed defective clonogenic ability and were incapable of repopulating the hematopoiesis of immunodeficient NSG mice. The development of DC-like hematopoietic stem cells will facilitate the understanding of the molecular and cellular basis of this inherited bone marrow failure syndrome, and will serve as a platform to evaluate the efficacy of new hematopoietic therapies for DC.

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Generation of dyskeratosis congenita-like hematopoietic stem cells through the stable inhibition of DKC1

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02145-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Carlos Carrascoso-Rubio ◽

Hidde A. Zittersteijn ◽

Laura Pintado-Berninches ◽

Beatriz Fernández-Varas ◽

M. Luz Lozano ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Failure ◽

Clinical Manifestations ◽

Dyskeratosis Congenita ◽

Bone Marrow Failure Syndrome ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Telomere Biology

AbstractDyskeratosis congenita (DC) is a rare telomere biology disorder, which results in different clinical manifestations, including severe bone marrow failure. To date, the only curative treatment for the bone marrow failure in DC patients is allogeneic hematopoietic stem cell transplantation. However, due to the toxicity associated to this treatment, improved therapies are recommended for DC patients. Here, we aimed at generating DC-like human hematopoietic stem cells in which the efficacy of innovative therapies could be investigated. Because X-linked DC is the most frequent form of the disease and is associated with an impaired expression of DKC1, we have generated DC-like hematopoietic stem cells based on the stable knock-down of DKC1 in human CD34+ cells with lentiviral vectors encoding for DKC1 short hairpin RNAs. At a molecular level, DKC1-interfered CD34+ cells showed a decreased expression of TERC, as well as a diminished telomerase activity and increased DNA damage, cell senescence, and apoptosis. Moreover, DKC1-interfered human CD34+ cells showed defective clonogenic ability and were incapable of repopulating the hematopoiesis of immunodeficient NSG mice. The development of DC-like hematopoietic stem cells will facilitate the understanding of the molecular and cellular basis of this inherited bone marrow failure syndrome and will serve as a platform to evaluate the efficacy of new hematopoietic therapies for DC.

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Generation of Dyskeratosis Congenita-like Hematopoietic Stem Cells through the Stable Inhibition of DKC1

10.21203/rs.3.rs-26927/v2 ◽

2020 ◽

Author(s):

Carlos Carrascoso-Rubio ◽

Hidde A. Zittersteijn ◽

Laura Pintado-Berninches ◽

Beatriz Fernández-Varas ◽

M. Luz Lozano ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Failure ◽

Clinical Manifestations ◽

Dyskeratosis Congenita ◽

Bone Marrow Failure Syndrome ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Telomere Biology

Abstract Dyskeratosis congenita (DC) is a rare telomere biology disorder, which results in different clinical manifestations, including severe bone marrow failure. To date, the only curative treatment for bone marrow failure in DC patients is allogeneic hematopoietic stem cell transplantation. However due to the toxicity associated to this treatment, improved therapies are recommended for DC patients. Here we aimed at generating DC-like human hematopoietic stem cells in which the efficacy of innovative therapies could be investigated. Because X-linked DC is the most frequent form of the disease and is associated with an impaired expression of DKC1, we have generated DC-like hematopoietic stem cells based on the stable knock-down of DKC1 in human CD34+ cells with lentiviral vectors encoding for DKC1 short hairpin RNAs. At a molecular level, DKC1-interfered CD34+ cells showed a decreased expression of TERC, as well as a diminished telomerase activity and increased DNA damage, cell senescence and apoptosis. Moreover, DKC1-interfered human CD34+ cells showed defective clonogenic ability and were incapable of repopulating the hematopoiesis of immunodeficient NSG mice. The development of DC-like hematopoietic stem cells will facilitate the understanding of the molecular and cellular basis of this inherited bone marrow failure syndrome, and will serve as a platform to evaluate the efficacy of new hematopoietic therapies for DC.

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Generation of Dyskeratosis Congenita-like Hematopoietic Stem Cells through the Stable Inhibition of DKC1

10.21203/rs.3.rs-26927/v1 ◽

2020 ◽

Author(s):

Carlos Carrascoso-Rubio ◽

Hidde A. Zittersteijn ◽

Laura Pintado-Berninches ◽

Beatriz Fernández-Varas ◽

M. Luz Lozano ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Hematopoietic Stem Cells ◽

Bone Marrow Failure ◽

Dyskeratosis Congenita ◽

Hematopoietic Stem ◽

Human Cd34

Abstract Dyskeratosis congenita is a rare telomere biology disorder, which results in different clinical manifestations, including severe bone marrow failure. To date, the only curative treatment for bone marrow failure in dyskeratosis congenita patients is allogeneic hematopoietic stem cell transplantation. However due to the toxicity associated to allogeneic hematopoietic stem cell transplantation in dyskeratosis congenita, new non-toxic therapies are recommended to improve the life expectancy of these patients. Since bone marrow biopsies are not routinely performed during the follow-up of dyskeratosis congenita patients, the availability of dyskeratosis congenita hematopoietic stem cells constitutes a major limitation in the development of new hematopoietic therapies for dyskeratosis congenita. Here we aimed at generating dyskeratosis congenita-like human hematopoietic stem cells in which the efficacy of new therapies could be investigated. X-linked dyskeratosis congenita is one of the most frequent variants of dyskeratosis congenita and is associated with an impaired expression of the DKC1 gene. In this study we thus generated dyskeratosis congenita-like hematopoietic stem cells based on the stable knock-down of DKC1 in human CD34+ cells, using lentiviral vectors encoding for DKC1 short hairpin RNAs. At a molecular level, DKC1-interfered CD34+ cells showed a decreased expression of TERC, as well as a diminished telomerase activity and increased DNA damage. Moreover, DKC1-interfered human CD34+ cells showed defective clonogenic ability and were incapable of repopulating the hematopoiesis of immunodeficient NSG mice. The development of dyskeratosis congenita-like hematopoietic stem cells will facilitate the understanding of the molecular and cellular basis of the bone marrow failure characteristic of dyskeratosis congenita patients, and will serve as a platform for the development of new hematopoietic therapies for dyskeratosis congenita patients.

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Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution

Blood ◽

10.1182/blood-2002-01-0025 ◽

2003 ◽

Vol 101 (1) ◽

pp. 112-118 ◽

Cited By ~ 71

Author(s):

Mo A. Dao ◽

Jesusa Arevalo ◽

Jan A. Nolta

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Surface ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells ◽

Cd34 Expression

Abstract The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45+/CD34− cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow–derived CD34+/CD38− stem/progenitor cells. The human CD45+ cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45+ cell were recovered from the marrow of bnx mice than from the marrow of human stem cell–engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45+/CD34− cells recovered from thebnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34+ progeny following retransplantation. These studies demonstrate that the human CD34+ population can act as a reservoir for generation of CD34− cells. In the current studies we demonstrate that human CD34+/CD38− cells can generate CD45+/CD34− progeny in a long-term xenograft model and that those CD45+/CD34− cells can regenerate CD34+ progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.

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Impact of Chemoselective Pressure on Integration Site Patterns of Lentivirally Transduced Human Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v112.11.4622.4622 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4622-4622

Author(s):

Nadja Grund ◽

Patrick Maier ◽

Uwe Appelt ◽

Heike Allgayer ◽

Frederik Wenz ◽

...

Keyword(s):

Stem Cells ◽

Gene Transfer ◽

Hematopoietic Stem Cells ◽

In Silico ◽

Integration Site ◽

General Distribution ◽

Cytostatic Drug ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells

Abstract Hematologic side effects of cancer chemotherapy like myelosuppression are frequently dose-limiting. Lentiviral gene therapy with cytostatic drug resistance gene transfer to human hematopoietic stem cells (CD34+) is a promising approach to overcome this problem. In this context it is of interest if chemotherapy mediated selection has an impact on lentiviral integration site patterns of transduced hematopoietic stem cells (CD34+). Concerning this issue, human CD34+ cells transduced with a lentiviral self-inactivating (SIN) vector encoding MGMTP140K (the O6-BG resistant mutant of O6-methylguanine- DNA methyltransferase) were in vitro treated with the alkylating agent BCNU. For integration site analysis LM-PCR was performed and integration patterns of the treated and untreated CD34+ cells were analyzed and compared with an in silico set of 106 random integrations. We found different integration preferences of the lentiviral vector between either the treated (82 integrations) or the untreated (30 integrations) CD34+ cells and the in silico set: both groups showed chromosomal preferences, a significant bias for integrations in genes (74,4% in the treated, respectively 70% in the untreated to 40% in the in silico group), especially by favouring introns, a random integration distribution regarding transcription start sites (TSS), and most importantly no significant differences concerning the number of integrations in or near cancer genes. Concerning all integration characteristics we could not find significant differences when comparing the untreated with the treated group. In conclusion, the general distribution of lentiviral integrations in either untreated or treated human CD34+ cells showed no distinct differences between both groups but significant differences compared to the in silico integration set. These results suggest that chemoselection of cells lentivirally overexpressing a specific chemoresistence gene might not influence the integration pattern. Therefore chemotherapy pressure seems not to hamper the safety of lentiviral vectors in gene transfer studies.

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Regulation of Hematopoietic Stem Cells by Interferons and by DNA Methylation: Implications for Bone Marrow Failure

Blood ◽

10.1182/blood.v124.21.sci-20.sci-20 ◽

2014 ◽

Vol 124 (21) ◽

pp. SCI-20-SCI-20

Author(s):

Margaret A. Goodell

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Bone Marrow Failure ◽

Stem Cell Differentiation ◽

Primary Myelofibrosis ◽

Differentiation Potential ◽

Hematopoietic Stem

Bone marrow failure (BMF), the inability to regenerate the differentiated cells of the blood, has a number of genetic and environmental etiologies, such as mutation of telomere-associated protein genes and immune-related aplastic anemia. Recently, mutations in DNA methyltransferase 3A (DNMT3A) have been found to be associated with approximately 15% of cases of primary myelofibrosis (MF), which can be a cause of BMF. The role of DNMT3A more broadly in hematopoiesis, and specifically in BMF, is currently poorly understood. DNMT3A is one of two de novo DNA methylation enzymes important in developmental fate choice. We showed that Dnmt3a is critical for normal murine hematopoiesis, as hematopoietic stem cells (HSCs) from Dnmt3a knockout (KO) mice displayed greatly diminished differentiation potential while their self-renewal ability was markedly increased1, in effect, leading to failure of blood regeneration or BMF. Combined with loss of Dnmt3b, HSCs exhibited a profound differentiation block, mediated in part by an increase of stabilized b-catenin. While we did not initially observe bone marrow pathology or malignancy development in mice transplanted with Dnmt3a KO HSCs, when we aged a large cohort of mice, all mice succumbed to hematologic disease within about 400 days. Roughly one-third of mice developed frank leukemia (acute lymphocytic leukemia or acute myeloid leukemia), one-third developed MDS, and the remainder developed primary myelofibrosis or chronic myelomonocytic leukemia. The pathological characteristics of the mice broadly mirror those of patients, suggesting the Dnmt3a KO mice can serve as a model for human DNMT3A-mutation associated disease. Strikingly, bone marrow of mice with different disease types exhibit distinct DNA methylation features. These will findings and the implications for disease development will be discussed. We are currently investigating the factors that drive different outcomes in the mice, including stressors such as exposure to interferons. We have hypothesized that HSC proliferation accelerates the Dnnmt3a-associated disease phenotypes. We have previously shown that interferons directly impinge on HSCs in the context of infections. Interferons activate HSCs to divide, generating differentiated progeny and cycling HSCs. Repeated interferon stimulation may permanently impair HSC function and bias stem cell output. When combined with loss of Dnmt3a, interferons may promote BMF. We will discuss broadly how external factors such as aging and infection may collaborate with specific genetic determinants to affect long-term hematopoiesis and malignancy development. Reference: Challen GA, Sun D, Jeong M, et al. Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet 2012; 44: 23-31 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Different kinases acting on stathmin (oncoprotein, Op18) in human healthy and malignant hematopoietic stem cells

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.17527 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 17527-17527

Author(s):

H. Lannert ◽

T. Able ◽

S. Leicht ◽

R. Saffrich ◽

V. Eckstein ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Expression Profiles ◽

Proteome Analysis ◽

Becton Dickinson ◽

Hematopoietic Stem ◽

Cd34 Cells

17527 Background: Stathmin/Op18 is a cytosolic phosphoprotein which regulates the dynamics of microtubules. This regulation is important in mitosis during cell division and in the migration of cells in modification of the cytoskeleton. The process of tumor proliferation and metastasis is characterized by high rates of mitosis and migration into distant tissues. Stathmin itself is regulated by kinases through phosphorylation of mainly 4 different serin sides. In this study, we investigated stathmin- and its kinases expression in native hematopoietic CD34+ stem cells (HSCs) from bone marrow (BM) in comparison to mobilized peripheral blood stem cells (mPBSCs) from G-CSF stimulated donors and leukemic CD34+ cells from patients with AML. Methods: Mononuclear cells were isolated by a standard Ficoll-Hypaque gradient separation method from the different blood sources. An Auto-MACS (Miltenyi) and FACS Vantage SE cell sorter (Becton Dickinson) was used to highly enrich (>99%) CD34+ cells fractions. In comparative proteome analysis, we detected the protein expression of stathmin in mPBSCs, AML CD34+ cells, and in native HSCs from BM. We performed microarray-based gene expression profiles of these cells and focused on kinases regulating stathmin’s activity. Furthermore, we monitored stathmin and its relevant kinases by FACS analyses of the enriched cell fractions and by fluorescence microscopy of bone marrow smears and cytospins. Results: In this study, we have shown in comparative proteome analysis (Q-TOF-MS/MS) that stathmin is expressed in G-CSF mobilized hematopoietic stem cells for the first time and in AML cells. In microarray analysis we indentified up- and down-regulated kinases: MAPK, PAK1, PKC beta/zeta, MEKK3 and CDKs. Accordingly, we demonstrated in FACS analyses and in immunofluorescence microscopy the high intracellular expression of PKCzeta in AML cells and MEKK3 as well PAK1 in mPBSCs. Conclusions: Our findings show that G-CSF stimulates Stathmin expression in mPBSCs and plays a key role in migration into peripheral blood. Furthermore, we show the different expression of kinases acting on stathmin in mPBSCs and AML cells. Consequently, stathmin and its relevant kinases promise to become a future target in therapies of malignant processes. No significant financial relationships to disclose.

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Cellular and molecular architecture of hematopoietic stem cells and progenitors in genetic models of bone marrow failure

JCI Insight ◽

10.1172/jci.insight.131018 ◽

2020 ◽

Vol 5 (4) ◽

Cited By ~ 1

Author(s):

Stephanie Heidemann ◽

Brian Bursic ◽

Sasan Zandi ◽

Hongbing Li ◽

Sagi Abelson ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Failure ◽

Genetic Models ◽

Molecular Architecture ◽

Hematopoietic Stem

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Ex Vivo Gene Therapy: Graft-versus-host Disease (GVHD) in NSG™ (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) Mice Transplanted with CD34+ Human Hematopoietic Stem Cells

Toxicologic Pathology ◽

10.1177/0192623319844484 ◽

2019 ◽

Vol 47 (5) ◽

pp. 656-660 ◽

Cited By ~ 3

Author(s):

Sundeep Chandra ◽

Patrizia Cristofori ◽

Carlos Fonck ◽

Charles A. O’Neill

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Graft Versus Host Disease ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Host Disease ◽

Graft Versus Host ◽

Human Cd34

A therapeutic option for monogenic disorders is gene therapy with ex vivo-transduced autologous hematopoietic stem cells (HSCs). Safety or efficacy studies of ex vivo-modified HSCs are conducted in humanized mouse models after ablation of the murine bone marrow and transfer of human CD34+ HSCs. Engrafted human CD34+ cells migrate to bone marrow and differentiate into various human hematopoietic lineages. A 12-week study was conducted in NSG™ mice to evaluate engraftment, differentiation, and safety of human CD34+ cells that were transduced ( ex vivo) with a proprietary lentiviral vector encoding a human gene (BMRN-1) or a mock (green fluorescent protein) vector. Several mice intravenously injected with naive CD34+ cells or transduced CD34+ cells had variable lymphohistiocytic inflammatory cell infiltrates and microgranulomas in the liver and lungs consistent with graft-versus-host disease (GVHD). Spleen, bone marrow, stomach, reproductive tract, but not the skin had similar inflammatory changes. Ex vivo viral transduction of CD34+ cells did not impact engraftment or predispose to xenogeneic GVHD.

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DNA Microarray Analysis of GPI-Negative Hematopoietic Stem Cells in Paroxysmal Nocturnal Hemoglobinuria Patients.

Blood ◽

10.1182/blood.v106.11.1048.1048 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1048-1048

Author(s):

Kazuhiko Ikeda ◽

Tsutomu Shichishima ◽

Yoshihiro Yamashita ◽

Yukio Maruyama ◽

Hiroyuki Mano

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Bone Marrow Failure ◽

Data Set ◽

Hematopoietic Stem ◽

Pnh Clone

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematological disorder which is manifested by complement-mediated hemolysis, venous thrombosis, and bone marrow failure. Deficiencies of glycosylphosphatidylinositol (GPI)-anchored proteins, due to mutations in the phosphatidylinositol glycan-class A (PIG-A) gene, contribute to complement-mediated hemolysis and affect all hematopoietic lineages in PNH. However, it is unclear how a PNH clone with a PIG-A gene mutation expands in bone marrow. Although some genes, including the Wilms’ tumor gene (Shichishima et al, Blood, 2002), the early growth response gene, anti-apoptosis genes, and the gene localized at breakpoints of chromosome 12, have been reported as candidate genes that may associate with proliferations of a GPI-negative PNH clone, previous studies were not intended for hematopoietic stem cell, indicating that the differences in gene expressions between GPI-negative PNH clones and GPI-positive cells from PNH patients remain unclear at the level of hematopoietic stem cell. To identify genes contributing to the expansion of a PNH clone, here we compared the gene expression profiles between GPI-negative and GPI-positive fractions among AC133-positive hematopoietic stem cells (HSCs). By using the FACSVantage (Becton Dickinson, San Jose, CA) cell sorting system, both of CD59+AC133+ and CD59− AC133+ cells were purified from bone marrow mononuclear cells obtained from 11 individuals with PNH. Total RNA was isolated from each specimen with the use of RNeasy Mini column (Qiagen, Valencia, CA). The mRNA fractions were amplified, and were used to generate biotin-labeled cDNAs by the Ovation Biotin system (NuGEN Technologies, San Carlos, CA). The resultant cDNAs were hybridized with a high-density oligonucleotide microarray (HGU133A; Affymetrix, Santa Clara, CA). A total of >22,000 probe sets (corresponding to >14,000 human genes) were assayed in each experiment, and thier expression intensities were analyzed by GeneSpring 7.0 software (Silicon Genetics, Redwood, CA). Comparison between CD59-negative and CD59-positive HSCs has identified a number of genes, expression level of which was statistically different (t-test, P <0.001) between the two fractions. Interestingly, one of the CD59− -specific genes isolated in our data set turned out to encode a key component of the proteasome complex. On the other hand, a set of transcriptional factors were specifically silenced in the CD59− HSCs. These data indicate that affected CD59-negative stem cells have a specific molecular signature which is distinct from that for the differentiation level-matched normal HSCs. Our data should pave a way toward the molecular understanding of PNH.

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