scholarly journals Distal-Less Homeobox 5 Is a Therapeutic Target for Attenuating Hypertrophy and Apoptosis of Mesenchymal Progenitor Cells

2020 ◽  
Vol 21 (14) ◽  
pp. 4823
Author(s):  
John Twomey-Kozak ◽  
Salomi Desai ◽  
Wenguang Liu ◽  
Neill Y. Li ◽  
Nicholas Lemme ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Knee Joint ◽  
Progenitor Cells ◽  
Apoptotic Cell ◽  
Cell Activity ◽  
Healthy Human ◽  
Human Cartilage ◽  
Mesenchymal Progenitors ◽  
Cell Hypertrophy ◽  
Human Knee Joint

Chondrocyte hypertrophy is a hallmark of osteoarthritis (OA) pathology. In the present study, we elucidated the mechanism underlying the relationship between the hypertrophy/apoptotic phenotype and OA pathogenesis in bone marrow-derived mesenchymal stem cells (BM-MSCs) via gene targeting of distal-less homeobox 5 (DLX5). Our primary objectives were (1) to determine whether DLX5 is a predictive biomarker of cellular hypertrophy in human osteoarthritic tissues; (2) To determine whether modulating DLX5 activity can regulate cell hypertrophy in mesenchymal stem/progenitor cells from marrow and cartilage. Whole transcriptome sequencing was performed to identify differences in the RNA expression profile between human-cartilage-derived mesenchymal progenitors (C-PCs) and bone-marrow-derived mesenchymal progenitors (BM-MSCs). Ingenuity Pathway Analysis (IPA) software was used to compare molecular pathways known to regulate hypertrophic terminal cell differentiation. RT-qPCR was used to measure DLX5 and hypertrophy marker COL10 in healthy human chondrocytes and OA chondrocytes. DLX5 was knocked down or overexpressed in BM-MSCs and C-PCs and RT-qPCR were used to measure the expression of hypertrophy/terminal differentiation markers following DLX5 modulation. Apoptotic cell activity was characterized by immunostaining for cleaved caspase 3/7. We demonstrate that DLX5 and downstream hypertrophy markers were significantly upregulated in BM-MSCs, relative to C-PCs. DLX5 and COL10 were also significantly upregulated in cells from OA knee joint tissues, relative to normal non-arthritic joint tissues. Knocking down DLX5 in BM-MSCs inhibited cell hypertrophy and apoptotic activity without attenuating their chondrogenic potential. Overexpression of DLX5 in C-PCs stimulated hypertrophy markers and increased apoptotic cell activity. Modulating DLX5 activity regulates cell hypertrophy and apoptosis in BM-MSCs and C-PCs. These findings suggest that DLX5 is a biomarker of OA changes in human knee joint tissues and confirms the DLX5 mechanism contributes to hypertrophy and apoptosis in BM-MSCs.

scholarly journals Development and Examination of a Knee Prosthesis Geometry

2019 ◽  
Vol 11 (1) ◽  
pp. 27-30
Author(s):  
Gábor Péter Balassa
Keyword(s):  
Knee Joint ◽  
Research Team ◽  
Knee Prosthesis ◽  
Experimental Measurements ◽  
Joint Movement ◽  
Knee Prostheses ◽  
Healthy Human ◽  
Human Knee ◽  
Human Knee Joint ◽  
Number Of Patients

Abstract The necessity for the knee prosthesis is confirmed by the large increase in the number of patients suffering from arthrosis, which is a present-day disease. Despite this need, there doesn’t exist an optimal knee prosthesis. Nowadays the development of the knee prostheses is progressing. It is very difficult to define the required geometry with traditional methods, because the movement conditions to be created by the prostheses should be similar to the movements of the human knee. During previous research the biomechanical research team of the Szent István University occupied with experimental measurements of the healthy human knee joint movement. In this paper I would like to introduce a method of prosthesis geometry development. As a result, a knee prosthesis geometry has been created which is approaching the movement form of the real human knee joint.

Diversity and Molecular Modeling of Neutrophil Elastase Mutations in Patients with Severe Congenital Neutropenia and Acute Myelogenous Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1454-1454
Author(s):  
Andrew A.G. Aprikyan ◽  
Steve Stein ◽  
Nara A. Markosyan ◽  
Maxim Totrov ◽  
Ruben Abagyan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Neutrophil Elastase ◽  
Apoptotic Cell ◽  
Myelogenous Leukemia ◽  
Congenital Neutropenia ◽  
Deletion Mutations ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells ◽  
Acute Myelogenous

Abstract Severe congenital neutropenia (SCN) is an inheritable hematopoietic disorder that is characterized by extremely low levels of neutrophils in peripheral circulation and maturation arrest of bone marrow myeloid progenitor cells at the promyelocytic stage of differentiation. SCN patients have recurring severe infections and approximately 10% of these patients evolve to develop acute myelogenous leukemia. Recently we reported that an impaired cell survival and cell cycle arrest of bone marrow myeloid progenitor cells was observed in SCN patients compared with controls. We also reported various heterozygous mutations in the neutrophil elastase (NE) gene encoding a serine protease in approximately 80% of SCN patients. We hypothesized that mutations in the NE gene trigger apoptotic cell death of myeloid progenitor cells and subsequent severe neutropenia. Mutational analysis of 15 families with one or more affected family members revealed that mutant NE was present only in affected but not in healthy members of these families suggesting the causative role for mutant NE in pathogenesis of SCN. Sequencing analysis revealed that none of SCN patients negative for NE mutations examined had mutations in the Gfi-1 or WAS gene. Sequencing DNA samples of SCN and SCN/AML patients revealed 40 mutations that are distributed primarily throughout the exons 2 through 5 of the NE gene and result in substitution, deletion, insertion, or truncation mutations. Molecular modeling of the tertiary structure of NE revealed that all these mutations can be grouped into three major categories. The first category includes 19 substitution and insertion mutations that are grouped around the N-glycosylation sites of the neutrophil elastase and may lead to abnormal targeting and subcellular localization of the mutant protease. The second group includes 9 substitution and deletion mutations that alter the side loop of the NE that is necessary for proper oligomerization of neutrophil elastase. The third category includes 12 substitution, truncation, and deletion mutations that either alter or completely eliminate the carboxy-terminus of the mutant protein leading to conformational changes of the binding pocket of the NE, and subsequently to altered substrate specificity and/or an acquired resistance to elastase inhibitors. SCN patients that evolved to develop AML had either substitution, deletion, or truncation mutations from each of the three categories described above. Most mutations are clearly non-conservative, have destabilizing effect on oligomeric structure of mutant protein, and alter dramatically the affinity of mutant NE to various factors participating in its processing and intracellular transport. Flow cytometry analysis of annexin V-labeled cells revealed that expression of representative mutant but not normal NE from each of the three categories of NE mutations in human promyelocytic HL-60 cells triggered apoptotic cell death similar to that observed in bone marrow progenitor cells in SCN patients. These data indicate that impaired cell survival and block of differentiation in SCN is due to heterozygous mutations in the neutrophil elastase gene. Current studies focused on design and screen of specific protease inhibitors capable of blocking the pro-apoptotic effect of mutant neutrophil elastase.

scholarly journals Delineation of T-progenitor cell activity within the CD34+ compartment of adult bone marrow

Blood ◽  
1995 ◽  
Vol 85 (10) ◽  
pp. 2770-2778 ◽  
Author(s):  
AH Galy ◽  
D Cen ◽  
M Travis ◽  
S Chen ◽  
BP Chen
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Cell Activity ◽  
Cell Potential ◽  
Hematopoietic Stem ◽  
Adult Bone Marrow ◽  
Hla Dr ◽  
Adult Bone

T-cell production is largely dependent on the presence of a thymus gland where CD34+ precursors mature into T lymphocytes. Prethymic stages of T-cell development are less defined. Therefore, this study aims to delineate T-progenitor cell potential within the CD34+ Lineage-- (Lin-) cell compartment of adult bone marrow (ABM). Fractionation of CD34+ Lin-ABM cells with CD45RA, Thy-1, CD38, and HLA-DR failed to absolutely segregate T-cell reconstituting ability, indicating broad distribution of T-progenitor cell potential. Titration experiments showed that low numbers of CD34+ Lin- CD45RA+ (RA+) cells had greater thymus repopulating ability than CD34+ Lin- CD45RA- cells (RA-). The great majority (> 95%) of RA+ cells expressed CD38, HLA-DR and 70% to 90% of RA+ cells lacked Thy-1 surface expression. RA+ cells contained colony-forming unit granulocyte-macrophage (CFU-GM) progenitor cells but were depleted of erythroid potential, did not provide hematopoietic reconstitution of human bone fragments implanted into SCID mice, and did not efficiently maintain CD34+ cells with secondary clonogenic potential in bone marrow cultures. Thus, RA+ cells are oligopotent (nonprimitive) CD34+ progenitors with T-cell reconstituting ability. In contrast, these same assays indicated that CD34+ Lin- CD45RA- cells (RA- cells) comprised hematopoietic stem cells (HSC) with primitive multilineage (T, B, myeloid, and erythroid) hematopoietic potential. It was confirmed that HSC-containing populations, such as CD34+ Lin- CD45RA- Thy-1+ cells had thymus repopulating ability. Culture of RA-cells on murine bone marrow stromal cells in the presence of interleukin (IL)-3, IL-6, and leukemia inhibitory factor (LIF) generated CD34+ CD45RA+ progeny engrafting in a secondary severe combined immunodeficiency (SCID)-hu thymus assay. Altogether, our results underscore the fact that T-cell reconstituting potential can be dissociated from HSC activity. Furthermore, we speculate that HSC might develop into the T lineage indirectly, via differentiation into an intermediate oligopotent CD34+ CD45RA+ stage. Finally, T-progenitor cells can be cultured in vitro.

Differential hMLH1 Gene Expression after Temozolomide Selection Linked to Microsatellite Instability in a Subset of Hematopoietic Stem/Progenitor Cells in Old Versus Young and Cancer Versus Normal Patient Samples.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 509-509
Author(s):  
Jonathan Kenyon ◽  
Emily Thomas ◽  
Karen Lingas ◽  
Stanton L. Gerson
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Microsatellite Instability ◽  
Progenitor Cells ◽  
Mismatch Repair ◽  
Dna Methyltransferase ◽  
Apoptotic Cell ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Age Related

Abstract The etiology of hematologic pathologies such as leukemia, myelodysplasia, anemia, bone marrow failure, altered immune function, and how they are associated with aging, remains unclear. Our hypothesis is that these diseases are caused or aggravated by a subset of hematopoietic stem/progenitor cells (HSC) lacking effective mismatch repair (MMR) and therefore exhibiting a hypermutator phenotype. Microsatellite instability (MSI) is a marker of MMR deficiency. We used cord blood, bone marrow, and bone core samples to isolate and then clonally expand HSC for MSI analysis. Five microsatellite loci previously used in the diagnosis of the MMR defective disease HNPCC (BAT 25, BAT 26, D2S123, D5S346, and D17S250) were analyzed for insertions and deletions. We have analyzed 38 patient samples between the ages of 0 and 86 years, including 8 cancer patients. These data show an age-dependent increase in the frequency of high grade microsatellite instability (MSI-H), i.e. those CFU with microsatellite instability at >20% of loci tested. Additionally, samples obtained from individuals older than 50 years were 6 times more likely to have a > 10% frequency of MSI-H CFU than samples obtained from younger individuals, suggesting an inflection point for the onset of hematopoietic diseases. In all instances this instability is seen only within a subset of human HSC clones. To further characterize the origin of this deficiency, a method to select for MMR deficient hematopoietic cells was developed that first selected for survival of MMR deficient HSC, and then allowed for the examination of expression status of key MMR pathway genes hMLH1 and hMSH2 and their protein products. First, CD34+ HSC were isolated from various aged patient samples. To avoid possible effects of other repair pathways, the cells were treated with O6-Benzylguanine (BG) to remove O6-methylguanine DNA-methyltransferase (MGMT) activity and prevent removal of O6-methylguanine lesions. Next, temozolomide (TMZ) at concentrations of 50–125 μM was used to induce O6-methylguanine (O6-mG) lesions that persist in the presence of BG. These O6-mG lesions mispair with cytosine and are recognized as DNA mismatches by the mismatch repair (MMR) pathway inducing apoptotic cell death. TMZ selected cells that fail to recognize the mispair due to a lack of MMR survive this selection. In these TMZ resistant clones, RT-PCR amplification of hMLH1 transcripts from total RNA isolated reveal a defect in hMLH1 but not hMSH2 expression. In the one AML sample obtained thus far HSC treated with 200 uM TMZ we have observed 0 to 30% of hMLH1 expression within TMZ resistant CFU was observed when compared to untreated controls. Together this data links MSI to MMR defects of a subpopulation of hematopoietic precursors in older individuals. This is the first examination of MMR gene expression in clones of HSC that has shown specific MMR functional deficiencies. Our study suggests that a MMR pathway deficiency in a subset of stem cells could contribute to age related hematopoietic disease processes including stem cell failure and malignant transformation.

Characterization of Clonotypic B Cell Progenitors in Bone Marrow of Multiple Myeloma Patients,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4002-4002
Author(s):  
Kelly Boucher ◽  
Nancy Parquet ◽  
Kenneth H. Shain ◽  
Rachid Baz ◽  
Melissa Alsina ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Stem Cell ◽  
B Cells ◽  
B Cell ◽  
Progenitor Cells ◽  
Research Funding ◽  
Cell Activity ◽  
Stem Cell Activity ◽  
B Cell Precursors

Abstract Abstract 4002 The lack of specific molecules to define malignant B progenitor cells in multiple myeloma (MM) has hampered the evaluation of minimal residual disease (MRD). We have identified a bone marrow (BM) CD138- subset that co-express CD19+ with identical κ or λ light chain (LC) restriction as the abnormal plasma cell (PC), as previously shown by others. The majority of LC restricted (LCR) B progenitors are CD19+/CD34- and <0.5% of whole BM (WBM) cells exhibit an immature phenotype: CD19+/CD34+ with aberrant CD27 expression. Immature B cell precursors are undetectable in peripheral blood (PB). LCR CD138-/CD19+ cells represent only 0.72± 0.5% of WBM in newly diagnosed patients (n=23) and do not increase (0.47± 0.51%) in patients with relapsed disease (n=21). The κ/λ LC ratio is 1.46±0.6 regardless of disease stage suggesting that conventional LC ratios for PCs (> 4 or <0.5) may not apply in B progenitors. LCR B progenitors (CD19+/34+ or CD19+/34-) are CD117+, Notch+ and Survivin+ as MM patient's hematopoietic stem cells (HSC). ALDH enzymatic activity is 3.1% (0.1-–7.26%) in LCR B cells. Flow sorted CD138+ did not grow in a colony formation assay (methylcellulose with PHA-LCM), whereas CD19+/CD34- or CD19+/CD34+ grew colonies with efficiency of 1:25,000 or 1:10000 respectively. Cells harvested from colonies have a lympho-plasmacytoid appearance and LCR B progenitors differentiated into CD138+ PC (80±5%) compared to HSC (10±5%). Colony efficiency was optimized (3 fold) using conditioned medium (CM) from HS5-stroma. Isolated CD138-/CD19+ cells were relatively bortezomib and melphalan resistant compared to CD138+ PC. We hypothesize that CD138-/CD19+/CD34+ cells contains earlier progenitor B cells that differentiate into the malignant PC. Surrogate assays for stem cell activity and xenotransplant models should determine cancer stem cell activity of immature B cell precursors. Research studies of MM putative progenitor cells will allow developing novel treatments to eradicate potential MM MRD reservoir. Disclosures: Baz: Millenium: Research Funding, Speakers Bureau; Celgene: Research Funding.

Protease inhibitors stimulate hematopoiesis and decrease apoptosis and ICE expression in CD34+ cells

Blood ◽  
2000 ◽  
Vol 96 (8) ◽  
pp. 2735-2739 ◽  
Author(s):  
Elaine M. Sloand ◽  
Jaroslaw Maciejewski ◽  
Princy Kumar ◽  
Sonnie Kim ◽  
Aniruddho Chaudhuri ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Hiv Infection ◽  
Progenitor Cells ◽  
Protease Inhibitors ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Apoptotic Cell ◽  
Hematologic Toxicity ◽  
Caspase 1 ◽  
Cd34 Cells

Highly active retroviral therapy has been associated with a decline in the frequency of cytopenia in patients with human immunodeficiency virus (HIV) infection. This may result from lower hematologic toxicity of newer antiviral drugs and their increased efficacy against HIV-1. Protease inhibitors, in addition to their effects on HIV replication, appear to affect various cellular functions. Recently, it was reported that ritonavir inhibited caspase-1 expression in normal CD4+ cells. It was hypothesized that protease inhibitors may improve hematopoietic function owing to their direct effects on the bone marrow progenitor cells. When ritonavir was added to methylcellulose cultures of bone marrow cells from HIV-infected patients and normal controls, colony formation increased 2.4-fold (n = 5) in control cultures and 4-fold (n = 5) in cultures of cells from HIV-infected patients. In the presence of ritonavir, cultures of CD34+ cells showed markedly decreased apoptosis in comparison with untreated cultures (45% decrease in apoptotic cell number; n = 6). A synthetic inhibitor of caspase 1 (Ac-Tyr-Val-Ala-Asp-aldehyde [single-letter amino acid codes]), which inhibits activation of several caspases including CPP32 and interleukin 1β–converting enzyme (ICE or caspase 1), also decreased the rate of apoptosis and enhanced colony formation by progenitor cells derived from HIV-infected patients (3-fold; n = 5). In ritonavir-treated samples derived from HIV-infected individuals, the number of cells expressing ICE also decreased. In conclusion, HIV protease inhibitors may, by blocking the caspase-dependent apoptotic pathway, overcome inhibition of hematopoiesis seen in patients with HIV infection, an effect unrelated to their antiviral activity.

Synergistic Induction of Apoptosis by Simultaneous Disruption of the Bcl-2 and mTOR/Akt Pathways in Acute Myeloid Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1588-1588
Author(s):  
Zhihong Zeng ◽  
Ismael Samudio ◽  
Michael Andreeff ◽  
Marina Konopleva
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Progenitor Cells ◽  
Apoptotic Cell ◽  
Mammalian Target Of Rapamycin ◽  
Leukemic Cells ◽  
Akt Phosphorylation ◽  
Combined Use ◽  
Frequent Event ◽  
Induced Apoptosis

Abstract Activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway provides survival signals for leukemic cells and blockade of this pathway may facilitate cell death. Akt/mTOR activation is a frequent event in AML that translates into significantly shorter survival of AML patients (S.M. Kornblau, ASH 2007). Bcl-2 family proteins are key regulators of apoptosis that are known to promote tumorigenesis and chemoresistance. We have recently reported that ABT-737, a small molecule BH3 mimetic, effectively kills AML progenitor cells via the disruption of Bcl-2:Bax, but its activity is largely diminished in cells overexpressing Mcl-1 (M. Konopleva, Cancer Cell 2006). We have now investigated anti-leukemia efficacy of concomitant blockade of mTOR/Akt, known to regulate Mcl-1 levels, and of Bcl-2 signaling. Combined use of rapamycin derivative temsirolimus and ABT-737 induced striking apoptotic responses in AML cell lines OCI-AML3 and MOLM13 leukemic cells. Mechanistically, temsirolimus downregulated expression of the anti-apoptotic protein Mcl-1 and induced expression levels of the proapoptotic BH3-only protein Noxa which specifically neutralizes Mcl-1. These effects were seen upon prolonged temsirolimus treatment that inhibited mTORC2 complex (Zeng, Blood 2007) and downregulated Akt phosphorylation. In primary AML samples co-cultured with bone marrow-derived stromal cells, ABT-737 at 25nM induced apoptotic cell death in CD34+ AML progenitor cells from 13 of the 16 samples tested. Temsirolimus/ABT-737 combination markedly enhanced apoptosis induction in 8/9 primary samples that were sensitive to temsirolimus alone (specific apoptosis, temsirolimus 15±5%; ABT-737, 28±5.9%; temsirolimus+ABT, 45±5.9%, p&lt;0.001) and in 3/7 samples resistant to temsirolimus (p=0.7). Further, combined blockade of mTOR/AKT and Bcl-2 pathway effectively induced apoptosis in 3 AML samples resistant to ABT-737 alone. Taken together, these results indicate that temsirolimus can directly alter the Bcl-2 apoptotic rheostat via induction of Noxa and decreased Mcl-1 expression sensitizing leukemia cells to apoptosis induced by BH3 mimetics. The combined use of agents interfering with AKT/mTOR and Bcl-2 pathways may be a highly effective anti-leukemia strategy that induces apoptosis in AML progenitor cells in the context of bone marrow microenvironment.

SSB1/NABP2 and SSB2/NABP1 Have Essential and Overlapping Roles in Maintaining Hematopoietic Stem and Progenitor Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2405-2405
Author(s):  
Wei Shi ◽  
Therese Vu ◽  
Glen Boyle ◽  
Fares Al-Ejeh ◽  
Tej Pandita ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Cell Death ◽  
Progenitor Cells ◽  
Apoptotic Cell ◽  
Genomic Stability ◽  
Apoptotic Cell Death ◽  
Interferon Response ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Single-stranded DNA binding (SSB) proteins are essential for a variety of DNA metabolic processes and the maintenance of genomic stability. SSB1 and its homolog SSB2, share greater sequence and domain homology to the archaeal and bacterial SSBs than eukaryotic RPA. They form complexes with two other proteins, C9Orf80 and INTS3, and play roles in mediating transcription and DNA repair. SSB1 (also known as OBFC2B or NABP2) is recurrently mutated in various cancers, however the precise function in normal development is incompletely understood. We have previously shown that Ssb1 is required for skeletogenesis, telomeric homeostasis and genomic stability in vivo while Ssb2 knockout mice are viable and grow normally without any detectable phenotype. Interestingly, we observed pronounced upregulation of Ssb2 in response to Ssb1 deletion and modest up-regulation of Ssb1 in response to Ssb2 deletion, suggesting that Ssb1 and Ssb2 may have some overlapping functions. To investigate the specific roles of both Ssb1 and Ssb2 in adult tissue homeostasis, we generated conditional double-knockout (DKO) mouse models of both genes. DKO in adult mice was achieved by using a tamoxifen-inducible Cre (Ssb1fl/fl Ssb2fl/fl R26-CreERT2), in which Ssb1 and Ssb2 are conditionally deleted by the administration of tamoxifen. Induced DKO mice become moribund within seven days featured with pancytopenia and dramatic loss of hematopoietic stem and progenitor cells (HSPCs), suggesting that Ssb1 and Ssb2 are required for the maintenance of haematopoietic stem and progenitors cells (HSPCs). DKO bone marrow was markedly hypocellular with reduction in all lineages of haematopoietic development. Functionally, HSPCs in DKO mice show decreased quiescence at the early stage followed by decreased proliferation and increased cell loss due to apoptotic cell death at the later stage, suggesting the imbalanced bone marrow homeostasis upon DKO may eventually result in exhaustion of the stem cell pool in DKO mice. Furthermore, bona fide HSPC intrinsic functional deficiency caused by DKO was confirmed by competitive bone marrow transplant, where DKO bone marrows showed abolished differentiation capacity and failed to repopulate the bone marrows of recipient mice after induction of DKO in the established engraftments from the Ssb1fl/fl Ssb2fl/fl R26-CreERT2 donors. Gene expression of DKO HSPCs demonstrated an exacerbated p53/p21 DNA damage response and pronounced interferon response. Validating these findings, stabilization of p53 and increased apoptotic cell death were observed in DKO bone marrows and HSPCs and induction of cell cycle and expression of interferon target genes was confirmed by QPCR. DKO HSPCs have increased expression of IFN induced surface markers such as Sca1. The IFN response was intrinsic to HSPCs. Mechanistically, DKO HSPCs manifest a profile of stalled replication forks on DNA combing analysis, unrepaired double strand breaks (increased gammaH2Ax foci and alkaline comet tail moment) and telomeric loss resulting in widespread chromosomal instability. DKO HSPC showed aberrant cytoplasmic accumulation of single stranded DNAs, with R-loop formation (DNA:RNA hybrid), driving this genetic instability and cell-intrinsic interferon response. Altogether, these data provide strong evidence that Ssb1 and Ssb2 have essential functions in regulating haematopoiesis through repairing replication associated DNA damage as well as resolution of R-loop generated during transcription, to maintain genomic stability during normal HSPC homeostasis. Disclosures No relevant conflicts of interest to declare.

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