A Novel Tissue and Stem Cell Specific TERF1 Splice Variant Is Downregulated in Tumour Cells

In this study, we describe the identification of a novel splice variant of TERF1/PIN2, one of the main components of the telomeric shelterin complex. This new splice variant is identical to TERF1, apart from a 30 amino acid internal insertion near to the C-terminus of TERF1. Based on genome comparison analyses and RNA expression data, we show that this splice variant is conserved among hominidae but absent from all other species. RNA expression and histological analyses show specific expression in human spermatogonial and hematopoietic stem cells (HSCs), while all other analyzed tissues lack the expression of this TERF1-isoform, hence the name TERF1-tsi (TERF1-tissue-specific-isoform). In addition, we could not detect any expression in primary human cells and established cancer cell lines. Immunohistochemistry results involving two new rabbit polyclonal antibodies, generated against TERF1-tsi specific peptides, indicate nuclear localization of TERF1-tsi in a subset of spermatogonial stem cells. In line with this observation, immunofluorescence analyzes in various cell lines consistently revealed that ectopic TERF1-tsi localizes to the cell nucleus, mainly but not exclusively at telomeres. In a first attempt to evaluate the impact of TERF1-tsi in the testis, we have tested its expression in normal testis samples versus matched tumor samples from the same patients. Both RT-PCR and IHC show a specific downregulation of TERF1-tsi in tumor samples while the expression of TERF1 and PIN2 remains unchanged.

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Microbiota Signals Suppress the Lymphoid Specification of Hematopoietic Stem Cells

Blood ◽

10.1182/blood-2021-151369 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 204-204

Author(s):

Joseph R. Krambs ◽

Darlene A. Monlish ◽

Feng Gao ◽

Laura G. Schuettpelz ◽

Daniel C. Link

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Expression Profiling ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Rna Expression ◽

Hematopoietic Stem ◽

Rna Expression Profiling ◽

The Impact

Abstract Aging is associated with an expansion of phenotypic hematopoietic stem cells (HSCs) with reduced self-renewal capacity and myeloid-skewed lineage differentiation. Signals from commensal flora support basal myelopoiesis in young mice; however, their contribution to hematopoietic aging is largely unknown. Here, we characterize hematopoiesis in young and middle-aged mice housed under specific pathogen free (SPF) and germ-free (GF) conditions. We did not analyze older mice due to the difficulty in maintaining mice in a gnotobiotic facility for more than one year. Consistent with prior studies, there is a shift in hematopoiesis in aged SPF mice towards granulopoiesis, with a significant increase in the percentage of granulocytic cells and a decrease in B lineage cells in the bone marrow. The marked shift from lymphopoiesis to myelopoiesis that develops during aging of SPF mice is mostly abrogated in GF mice. Compared with aged SFP mice, there is a marked expansion of B lymphopoiesis in aged GF mice, which is evident at the earliest stages of B cell development. To investigate the impact of microbiota signals on multipotent HSPCs, we first quantified HSPCs by flow cytometry (Figure 1A-B). In aged SPF mice, the number of lineage - Sca1 + cKit + CD150 + CD48 - (LSK-SLAM) cells and CD34 - LSK-SLAM cells is increased 6.4 ± 1.7-fold and 3.4 ± 1.2-fold, respectively. Similar increases were observed in aged GF mice, with LSK-SLAM increasing 5.3 ± 1.6-fold (p=NS compared to SPF mice) and CD34 - LSK-SLAM cells increasing 2.8 ± 0.31-fold (p=NS). To quantify functional HSCs, limiting dilution transplantation experiments using unsorted bone marrow cells was performed. Although on a per cell basis the repopulating activity of aged HSCs is reduced, due to the large increase in phenotypic HSCs, the number of functional HSCs actually increases with aging, with similar increases in functional HSCs in aged SPF and GF mice (Figure 1C). Finally, to assess lineage-bias, we transplanted a limiting number of sorted HSCs and assessed lineage output. As expected, in young SPF mice, the majority of HSCs displayed a balanced myeloid/lymphoid lineage output, with a significant increase in myeloid-biased HSCs observed with aging (Figure 1D). In young GF mice, the majority of HSCs are lymphoid-biased. Moreover, although the myeloid output increased modestly with aging, the majority of HSCs in aged GF remained lymphoid-biased or balanced. Consistent with these data, RNA expression profiling of phenotypic HSCs from aged GF mice show enrichment for non-myeloid biased HSCs. Surprisingly, the RNA expression profiling data also suggest that inflammatory signaling is increased in aged GF HSCs compared with aged SPF HSCs. Collectively, these data suggest that microbiota-related signals suppress the lymphoid potential of HSCs, contributing to the expansion of myeloid-biased HSCs that occurs with aging. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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The Impact of Age and Gender on Hematopoietic Stem Cells and Immune Contexture of the Bone Marrow Microenvironment

Cells Tissues Organs ◽

10.1159/000510774 ◽

2020 ◽

pp. 1-6

Author(s):

Rebar N. Mohammed

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Immune Cells ◽

Absolute Number ◽

Cell Number ◽

Hematopoietic Stem ◽

The Absolute ◽

And Gender ◽

The Impact

Hematopoietic stem cells (HSCs) are a rare population of cells that reside mainly in the bone marrow and are capable of generating and fulfilling the entire hematopoietic system upon differentiation. Thirty-six healthy donors, attending the HSCT center to donate their bone marrow, were categorized according to their age into child (0–12 years), adolescence (13–18 years), and adult (19–59 years) groups, and gender into male and female groups. Then, the absolute number of HSCs and mature immune cells in their harvested bone marrow was investigated. Here, we report that the absolute cell number can vary considerably based on the age of the healthy donor, and the number of both HSCs and immune cells declines with advancing age. The gender of the donor (male or female) did not have any impact on the number of the HSCs and immune cells in the bone marrow. In conclusion, since the number of HSCs plays a pivotal role in the clinical outcome of allogeneic HSC transplantations, identifying a younger donor regardless the gender is critical.

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A Novel, Myeloid Transcription Factor, C/EBPε, Is Upregulated During Granulocytic, But Not Monocytic, Differentiation

Blood ◽

10.1182/blood.v90.7.2591.2591_2591_2600 ◽

1997 ◽

Vol 90 (7) ◽

pp. 2591-2600 ◽

Cited By ~ 9

Author(s):

Roberta Morosetti ◽

Dorothy J. Park ◽

Alexey M. Chumakov ◽

Isabelle Grillier ◽

Masaaki Shiohara ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Hematopoietic Stem Cells ◽

Cell Line ◽

Cell Lines ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Nb4 Cells

Human C/EBPε is a newly cloned CCAAT/enhancer-binding transcription factor. Initial studies indicated it may be an important regulator of human myelopoiesis. To elucidate the range of expression of C/EBPε, we used reverse transcription-polymerase chain reaction (RT-PCR) analysis and examined its expression in 28 hematopoietic and 14 nonhematopoietic cell lines, 16 fresh myeloid leukemia samples, and normal human hematopoietic stem cells and their mature progeny. Prominent expression of C/EBPε mRNA occurred in the late myeloblastic and promyelocytic cell lines (NB4, HL60, GFD8), the myelomonoblastic cell lines (U937 and THP-1), the early myeloblast cell lines (ML1, KCL22, MDS92), and the T-cell lymphoblastic leukemia cell lines CEM and HSB-2. For the acute promyelocytic leukemia cell line NB4, C/EBPε was the only C/EBP family member that was easily detected by RT-PCR. No C/EBPε mRNA was found in erythroid, megakaryocyte, basophil, B lymphoid, or nonhematopoietic cell lines. Most acute myeloid leukemia samples (11 of 12) from patients expressed C/EBPε. Northern blot and RT-PCR analyses showed that C/EBPε mRNA decreased when the HL60 and KG-1 myeloblast cell lines were induced to differentiate toward macrophages. Similarly, Western blot analysis showed that expression of C/EBPε protein was either unchanged or decreased slightly as the promyelocytic cell line NB4 differentiated down the macrophage-like pathway after treatment with a potent vitamin D3 analog (KH1060). In contrast, C/EBPε protein levels increased dramatically as NB4 cells were induced to differentiate down the granulocytic pathway after exposure to 9-cis retinoic acid. Furthermore, very early, normal hematopoietic stem cells (CD34+/CD38−), purified from humans had very weak expression of C/EBPε mRNA, but levels increased as these cells differentiated towards granulocytes. Likewise, purified granulocytes appeared to express higher levels of C/EBPε mRNA than purified macrophages. Addition of phosphothiolated antisense, but not sense oligonucleotides to C/EBPε, decreased clonal growth of HL-60 and NB4 cells by about 50% compared with control cultures. Taken together, our results indicate that expression of C/EBPε is restricted to hematopoietic tissues, especially myeloid cells as they differentiate towards granulocytes and inhibition of its expression in HL-60 and NB4 myeloblasts and promyelocytes decreased their proliferative capacity. Therefore, this transcriptional factor may play an important role in the process of normal myeloid development.

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Dissecting the Immune Cell Progeny of CAR-Expressing Hematopoietic Stem Cells in a Humanized Mouse Model

Blood ◽

10.1182/blood-2019-129746 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4640-4640

Author(s):

Heng-Yi Liu ◽

Nezia Rahman ◽

Tzu-Ting Chiou ◽

Satiro N. De Oliveira

Keyword(s):

Stem Cells ◽

T Cells ◽

T Cell ◽

Hematopoietic Stem Cells ◽

Cell Lines ◽

Immune Cells ◽

Research Funding ◽

Humanized Mice ◽

Tumor Challenge ◽

Hematopoietic Stem

Background: Chemotherapy-refractory or recurrent B-lineage leukemias and lymphomas yield less than 50% of chance of cure. Therapy with autologous T-cells expressing chimeric antigen receptors (CAR) have led to complete remissions, but the effector cells may not persist, limiting clinical efficacy. Our hypothesis is the modification of hematopoietic stem cells (HSC) with anti-CD19 CAR will lead to persistent generation of multilineage target-specific immune cells, enhancing graft-versus-cancer activity and leading to development of immunological memory. Design/Methods: We generated second-generation CD28- and 4-1BB-costimulated CD19-specific CAR constructs using third-generation lentiviral vectors for modification of human HSC for assessment in vivo in NSG mice engrafted neonatally with human CD34-positive cells. Cells were harvested from bone marrows, spleens, thymus and peripheral blood at different time points for evaluation by flow cytometry and ddPCR for vector copy numbers. Cohorts of mice received tumor challenge with subcutaneous injection of lymphoma cell lines. Results: Gene modification of HSC with CD19-specific CAR did not impair differentiation or proliferation in humanized mice, leading to CAR-expressing cell progeny in myeloid, NK and T-cells. Humanized NSG engrafted with CAR-modified HSC presented similar humanization rates to non-modified HSC, with multilineage CAR-expressing cells present in all tissues with stable levels up to 44 weeks post-transplant. No animals engrafted with CAR-modified HSC presented autoimmunity or inflammation. T-cell populations were identified at higher rates in humanized mice with CAR-modified HSC in comparison to mice engrafted with non-modified HSC. CAR-modified HSC led to development of T-cell effector memory and T-cell central memory phenotypes, confirming the development of long-lasting phenotypes due to directed antigen specificity. Mice engrafted with CAR-modified HSC successfully presented tumor growth inhibition and survival advantage at tumor challenge with lymphoma cell lines, with no difference between both constructs (62.5% survival for CD28-costimulated CAR and 66.6% for 41BB-costimulated CAR). In mice sacrificed due to tumor development, survival post-tumor injection was directly correlated with tumor infiltration by CAR T-cells. Conclusions: CAR modification of human HSC for cancer immunotherapy is feasible and continuously generates CAR-bearing cells in multiple lineages of immune cells. Targeting of different malignancies can be achieved by adjusting target specificity, and this approach can augment the anti-lymphoma activity in autologous HSC recipients. It bears decreased morbidity and mortality and offers alternative therapeutic approach for patients with no available sources for allogeneic transplantation, benefiting ethnic minorities. Disclosures De Oliveira: National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London: Research Funding; NIAID, NHI: Research Funding; Medical Research Council: Research Funding; CIRM: Research Funding; National Gene Vector Repository: Research Funding.

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Autophagy, a key mechanism of oncogenesis and resistance in leukemia

Blood ◽

10.1182/blood-2016-07-692707 ◽

2017 ◽

Vol 129 (5) ◽

pp. 547-552 ◽

Cited By ~ 55

Author(s):

Patrick Auberger ◽

Alexandre Puissant

Keyword(s):

Stem Cells ◽

Leukemic Cell ◽

Degenerative Diseases ◽

Leukemic Transformation ◽

Adaptation Mechanism ◽

Hematopoietic Stem ◽

The Past ◽

Response To Chemotherapy ◽

Cancer Initiation ◽

The Impact

AbstractAutophagy is a lysosomal pathway involved in degradation of intracellular material. It appears as an adaptation mechanism that is essential for cellular homeostasis in response to various stress conditions. Over the past decade, many studies have linked alteration of autophagy with cancer initiation and progression, autoimmune, inflammatory, metabolic, and degenerative diseases. This review highlights recent findings on the impact of autophagy on leukemic transformation of normal hematopoietic stem cells and summarizes its role on leukemic cell response to chemotherapy.

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Lentivirus-based vectors transduce mouse hematopoietic stem cells with similar efficiency to Moloney murine leukemia virus–based vectors

Blood ◽

10.1182/blood.v96.10.3385 ◽

2000 ◽

Vol 96 (10) ◽

pp. 3385-3391 ◽

Cited By ~ 31

Author(s):

Stephane Barrette ◽

Janet L. Douglas ◽

Nancy E. Seidel ◽

David M. Bodine

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Lines ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Moloney Murine Leukemia Virus ◽

Lentivirus Vector ◽

Hematopoietic Stem ◽

Lentivirus Vectors ◽

Murine Leukemia

Abstract The low levels of transduction of human hematopoietic stem cells (HSCs) with Moloney murine leukemia virus (MLV) vectors have been an obstacle to gene therapy for hematopoietic diseases. It has been demonstrated that lentivirus vectors are more efficient than MLV vectors at transducing nondividing cell lines as well as human CD34+ cells and severe combined immunodeficiency disease repopulating cells. We compared transduction of cell lines and Lin− bone marrow cells, using a vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus or MLV vectors carrying a green fluorescent protein marker gene. As predicted, the lentivirus vector was more efficient at transducing mouse and human growth-inhibited cell lines. The transduction of mouse HSC by lentivirus vectors was compared directly to MLV vectors in a co-transduction assay. In this assay, transduction by ecotropic MLV is a positive internal control for downstream steps in retrovirus transduction, including cell division. Both the VSV-G lentivirus and MLV vectors transduced mouse HSCs maintained in cytokine-free medium at very low frequency, as did the ecotropic control. The lentivirus vector and the MLV vector were equally efficient at transducing bone marrow HSCs cultured in interleukin 3 (IL-3), IL-6, and stem cell factor for 96 hours. In conclusion, although lentivirus vectors are able to transduce growth-inhibited cell lines, the cell cycle status of HSCs render them resistant to lentivirus-mediated transduction, and it is hypothesized that entry into cycle, not necessarily division, may be a requirement for efficient lentivirus-mediated transduction.

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Regulatory Roles of Bone in Neurodegenerative Diseases

Frontiers in Aging Neuroscience ◽

10.3389/fnagi.2020.610581 ◽

2020 ◽

Vol 12 ◽

Author(s):

Zhengran Yu ◽

Zemin Ling ◽

Lin Lu ◽

Jin Zhao ◽

Xiang Chen ◽

...

Keyword(s):

Stem Cells ◽

Neurodegenerative Diseases ◽

The Elderly ◽

Lymphoid Organ ◽

The Body ◽

Hematopoietic Stem ◽

Brain Functions ◽

And Function ◽

The Impact ◽

The Brain

Osteoporosis and neurodegenerative diseases are two kinds of common disorders of the elderly, which often co-occur. Previous studies have shown the skeletal and central nervous systems are closely related to pathophysiology. As the main structural scaffold of the body, the bone is also a reservoir for stem cells, a primary lymphoid organ, and an important endocrine organ. It can interact with the brain through various bone-derived cells, mostly the mesenchymal and hematopoietic stem cells (HSCs). The bone marrow is also a place for generating immune cells, which could greatly influence brain functions. Finally, the proteins secreted by bones (osteokines) also play important roles in the growth and function of the brain. This article reviews the latest research studying the impact of bone-derived cells, bone-controlled immune system, and bone-secreted proteins on the brain, and evaluates how these factors are implicated in the progress of neurodegenerative diseases and their potential use in the diagnosis and treatment of these diseases.

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Critical windows in development of the rodent immune system

Human & Experimental Toxicology ◽

10.1191/0960327102ht287oa ◽

2002 ◽

Vol 21 (9-10) ◽

pp. 493-498 ◽

Cited By ~ 85

Author(s):

K S Landreth

Keyword(s):

Stem Cells ◽

Immune System ◽

Chemical Exposure ◽

Anatomical Location ◽

Hematopoietic Stem ◽

Windows Of Vulnerability ◽

The Impact ◽

Timing Of Exposure ◽

Developmental Immunotoxicity ◽

Developmental Windows

The immune system of rodents, like that in humans, develops from a population of pluripotential hematopoietic stem cells (HSC) that are generated early in gestation from uncommitted mesenchymal stem cells in the intra-embryonic splanchnoplure surrounding the heart. This early population of HSC gives rise to all circulating blood cell lineages, including cells of the innate and acquired immune system. To access the impact of chemical exposure on the developing immune system and establish developmental windows of potential vulnerability to these exposures, it is essential to first consider the anatomical development of hematopoietic and lymphopoietic tissues and the sequence of appearance of cells that give rise to the immune system. This is particularly true in embryonic development because, after they initially appear in intra-embryonic mesenchyme early in gestation, HSC migrate through an orderly series of tissues before establishing residence in the bone marrow and thymus. The effect of exposure to chemical insults in utero, then, may differ depending on the specific timing of exposure and anatomical location of hematopoiesis. Mechanisms and consequences of developmental immunotoxicity in experimental animals will need to be considered in that context. This review presents an overview of developmental hematopoiesis and a working hypothesis of critical developmental windows of vulnerability of this developmental system to toxic insult by chemical exposure.

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High-level ectopic HOXB4 expression confers a profound in vivo competitive growth advantage on human cord blood CD34+ cells, but impairs lymphomyeloid differentiation

Blood ◽

10.1182/blood-2002-03-0767 ◽

2003 ◽

Vol 101 (5) ◽

pp. 1759-1768 ◽

Cited By ~ 110

Author(s):

Bernhard Schiedlmeier ◽

Hannes Klump ◽

Elke Will ◽

Gökhan Arman-Kalcek ◽

Zhixiong Li ◽

...

Keyword(s):

Stem Cells ◽

Cord Blood ◽

Therapeutic Window ◽

Hematopoietic Stem ◽

Expression Levels ◽

Cd34 Cells ◽

Cord Blood Cd34 ◽

The Impact

Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However, HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs, we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34+ cells together with green fluorescent protein (GFP) as a reporter protein, and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic–severe combined immunodeficient (NOD/SCID) mice or in competition with control vector–transduced cells, HOXB4-overexpressing cord blood CD34+ cells had a selective growth advantage in vivo, which resulted in a marked enhancement of the primitive CD34+ subpopulation (P = .01). However, high HOXB4 expression substantially impaired the myeloerythroid differentiation program, and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P < .03) and in vivo (P = .01). Furthermore, HOXB4 overexpression also significantly reduced B-cell output (P < .01). These results show for the first time unwanted side effects of ectopic HOXB4 expression and therefore underscore the need to carefully determine the therapeutic window of HOXB4 expression levels before initializing clinical trials.

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Kinetics of normal hematopoietic stem and progenitor cells in a Notch1-induced leukemia model

Blood ◽

10.1182/blood-2009-06-227843 ◽

2009 ◽

Vol 114 (18) ◽

pp. 3783-3792 ◽

Cited By ~ 41

Author(s):

Xiaoxia Hu ◽

Hongmei Shen ◽

Chen Tian ◽

Hui Yu ◽

Guoguang Zheng ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Quiescent State ◽

Hematopoietic Stem ◽

Definitive Evidence ◽

Accelerated Proliferation ◽

Stem And Progenitor Cells ◽

Underlying Mechanisms ◽

The Impact

Abstract The predominant outgrowth of malignant cells over their normal counterparts in a given tissue is a shared feature for all types of cancer. However, the impact of a cancer environment on normal tissue stem and progenitor cells has not been thoroughly investigated. We began to address this important issue by studying the kinetics and functions of hematopoietic stem and progenitor cells in mice with Notch1-induced leukemia. Although hematopoiesis was progressively suppressed during leukemia development, the leukemic environment imposed distinct effects on hematopoietic stem and progenitor cells, thereby resulting in different outcomes. The normal hematopoietic stem cells in leukemic mice were kept in a more quiescent state but remained highly functional on transplantation to nonleukemic recipients. In contrast, the normal hematopoietic progenitor cells in leukemic mice demonstrated accelerated proliferation and exhaustion. Subsequent analyses on multiple cell-cycle parameters and known regulators (such as p21, p27, and p18) further support this paradigm. Therefore, our current study provides definitive evidence and plausible underlying mechanisms for hematopoietic disruption but reversible inhibition of normal hematopoietic stem cells in a leukemic environment. It may also have important implications for cancer prevention and treatment in general.

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