Number Of Hematopoietic Stem Cells Research Articles

Chronic inflammation decreases HSC fitness by activating the druggable Jak/Stat3 signaling pathway

Chronic inflammation represents a major threat to human health since long‐term systemic inflammation is known to affect distinct tissues and organs. Recently, solid evidence demonstrated that chronic inflammation affects hematopoiesis; however, how chronic inflammation affects hematopoietic stem cells (HSCs) on the mechanistic level is poorly understood. Here, we employ a mouse model of chronic multifocal osteomyelitis (CMO) to assess the effects of a spontaneously developed inflammatory condition on HSCs. We demonstrate that hematopoietic and nonhematopoietic compartments in CMO BM contribute to HSC expansion and impair their function. Remarkably, our results suggest that the typical features of murine multifocal osteomyelitis and the HSC phenotype are mechanistically decoupled. We show that the CMO environment imprints a myeloid gene signature and imposes a pro‐inflammatory profile on HSCs. We identify IL‐6 and the Jak/Stat3 signaling pathway as critical mediators. However, while IL‐6 and Stat3 blockage reduce HSC numbers in CMO mice, only inhibition of Stat3 activity significantly rescues their fitness. Our data emphasize the detrimental effects of chronic inflammation on stem cell function, opening new venues for treatment.

The features of tumor niche cell composition in invasive breast ductal carcinoma of no special type

Breast cancer is the most common cancer and the leading cause of cancer death in woman of childbearing age. Tumor progression depends on the character of stromal-parenchymal interactions. Tumor microenvironment exerts a key influence on tumor progression. Tumor niche is an important element of the tumor microenvironment. According to existing ideas, tumor niche consists on immune cells and bone marrow progenitor cells. The present study describes the parameters of tumor niche in invasive breast carcinoma of no special type (IC-NST), associated with lymph node metastases. The purpose of the study was to investigate the features of tumor niche cell composition in IC-NST. Material and methods. The study included 128 patients with IC-NST (T1–3N0–3M0), who underwent total mastectomy or partial mastectomy with axillary lymph node dissection. The age of the patients ranged from 29 to 90. Histological examination of surgical specimens was carried out in accordance with standard methods. Suspensions of fresh frozen tumor surgical specimens were prepared for the hematopoetic progenitor cells identification. The antibodies against CD34, CD133, CD90, CD11B, CD45, AND CD202 were applied. Results. The study showed that the total number of hematopoietic stem and progenitor cells and macrophage progenitor cells in an amount exceeding 1.24 cells per 100 tumor cells was associated with the risk of developing lymph node metastases and large tumor size. Conclusion. The results obtained may be useful for understanding the role of tumor niche in tumor growth and lymph node metastasis of IC-NST.

Excessive physical exercise caused a decrease in CD4+ and Hematopoietic Stem Cell (HSC) levels in Wistar rats

Background: Excessive physical exercise can cause injury and disrupt the body's homeostasis. Excessive physical exercise also causes increasing oxygen demand and reactive oxygen species production (ROS) that induce damage in various cells and body tissue, including HSC and CD4+ cells. This study aims to prove that excessive physical exercise decreases the CD4+ and HSC levels in Wistar rats and evaluates the correlation between HSC and CD4+ cell levels. Methods: An experimental study with a randomized post-test-only control group design was conducted among 44 male Wistar rats in Clinical Pathology Laboratory, Mangusada General Hospital, Bali, from June to September 2022. The samples were divided into two groups; 22 rats received excessive physical exercise (treatment group) and 22 rats as the control group. The CD4+ cells and HSC were examined after 14 days of intervention. Data regarding the means of CD4+ and HSC levels between the two groups were assessed, and the correlation between HSC and CD4+ cell levels was analyzed using SPSS version 21 for Windows. Results: We used 22 rats for each treatment and control group. The mean level of CD4+ and HSC were significantly lower in the treatment group compared to the control group (356.41 vs. 661.41x106 cell/µl; p<0.001) and (359.14 vs. 551.09 x106 cell/µl). We found a statistically significant difference in CD4+ and HSC levels between the two groups, p<0.001). We also found a positive correlation between HSC and CD4+ level with r=0.636; p<0.001. Excessive physical exercise affected the immune function by decreasing CD4+ ad HSC levels in Wistar rats. Conclusions: The impact of excessive physical exercise on immune function showed by decreasing the level of CD4+ and HSC level in Wistar rats. The HSC number also has a positive correlation with CD4+ cell level.

CT-314 Multicenter Long-Term Follow Up of Allogeneic Hematopoietic Stem Cell Transplantation With Omidubicel: A Pooled Analysis of Five Prospective Clinical Trials

Allogeneic hematopoietic stem cell transplantation (allo-HCT) with umbilical cord blood (UCB) is limited by the low number of hematopoietic stem cells. Omidubicel is an ex-vivo expanded stem cell product derived from UCB. Prospective clinical trials have demonstrated faster engraftment and fewer infections with omidubicel compared to UCB, but long-term outcomes are unknown. This is a pre-specified pooled secondary analysis of long-term outcomes with allo-HCT using omidubicel from 5 multicenter prospective trials. Patients transplanted with omidubicel in one randomized phase III and four single-arm trials between 2006 and 2020 were followed for up to 10 years post-transplant. Twenty-six international academic transplant centers. Among 116 patients transplanted with either standalone omidubicel (n=92) or omidubicel with supplementary UCB (n=24), 97 (83.6%) engrafted with omidubicel, 11 (9.5%) with UCB, 2 mixed chimerism, 1 unevaluable, and 5 (4.3%) primary graft failure. This study included all patients aside from those engrafted with UCB (n=105). Median age was 42 (range, 2-62), 52.4% male, 30.5% non-white, and 30.5% high/very-high disease risk indices. Most common diseases were AML (40.1%), ALL (26.7%), MDS (13.3%), and sickle cell disease (7.6%). Median follow-up was 22.0 months (range, 0.3-122.5). The 3-year OS and DFS were 62.5% (95%CI, 53.4-73.2) and 56.2% (95%CI, 47.0-67.1), respectively. Common causes of death included disease relapse (n=16) and infection (n=11). Three-year cumulative incidences of chronic GVHD and relapse were 37.8% (95%CI, 27.9-47.6) and 24.3% (95%CI, 16.1-33.3), respectively. Durable trilineage hematopoiesis was observed for up to 10 years. Similarly, median numbers of lymphoid subsets, including CD3+, CD4+, CD8+ T cells, CD19+ B cells, and CD16+/CD56+ NK cells were maintained within normal range for up to 8 years. Secondary graft failure was noted in 5 patients, 3 of whom underwent a second allo-HCT. Secondary hematologic malignancies included donor-derived myeloid neoplasm (dd-MN) (n=1; 40 months post-transplant) and PTLD (n=2; 17 and 20 months), with one death attributed to PTLD. Notably, there was also one case of dd-MN in the control UCB group of the phase III study. Omidubicel demonstrated durable long-term hematopoiesis and immune competence. One case of dd-MN was observed with both omidubicel and control UCB.

Murine foetal liver supports limited detectable expansion of life-long haematopoietic progenitors.

Current dogma asserts that the foetal liver (FL) is an expansion niche for recently specified haematopoietic stem cells (HSCs) during ontogeny. Indeed, between embryonic day of development (E)12.5 and E14.5, the number of transplantable HSCs in the murine FL expands from 50 to about 1,000. Here we used a non-invasive, multi-colour lineage tracing strategy to interrogate the embryonic expansion of murine haematopoietic progenitors destined to contribute to the adult HSC pool. Our data show that this pool of fated progenitors expands only two-fold during FL ontogeny. Although Histone2B-GFP retention in vivo experiments confirmed substantial proliferation of phenotypic FL-HSC between E12.5 and E14.5, paired-daughter cell assays revealed that many mid-gestation phenotypic FL-HSCs are biased to differentiate, rather than self-renew, relative to phenotypic neonatal and adult bone marrow HSCs. In total, these data support a model in which the FL-HSC pool fated to contribute to adult blood expands only modestly during ontogeny.

COMPARISON OF THE PROLIFERATIVE RESPONSES OF HEMATOPOIETIC STEM CELLS EXPOSED TO LOW DOSE RATE RADIATION IN VIVO AND EX VIVO.

The hematopoietic stem cells (HSCs) are sensitive to radiation. Chronic exposure to low dose rate (LDR) radiation at 20mGy/day results in a decrease in the number of HSCs and an increase of leukemia. In this study, the proliferative capacities of ex vivo HSCs, exposed to 20mGy/day of gamma-rays for 20days, were compared with those of in vivo HSCs from similarly whole-body-irradiated mice. Radiation suppressed the growth of the ex vivo HSCs after Day 16 of irradiation and until Day 7 post-exposure. Almost all types of cells, particularly multipotent progenitors, common myeloid progenitors, granulocytes and macrophages, were significantly reduced in number at Day 20 of irradiation and Day 7 post-exposure in culture. HSCs and multipotent progenitors irradiated in vivo, however, decreased transiently and recovered by Day 7 post-exposure. These findings suggest that the microenvironment in vivo protects HSCs from the effects of LDR radiation.

Frizzled-6 promotes hematopoietic stem/progenitor cell mobilization and survival during LPS-induced emergency myelopoiesis

SummaryEmergency hematopoiesis involves the activation of bone marrow hematopoietic stem/progenitor cells (HSPCs) in response to systemic inflammation by a combination of cell-autonomous and stroma-dependent signals and leads to their release from bone marrow and migration to periphery. We have previously shown that FZD6 plays a pivotal role in regulating HSPC expansion and long-term maintenance. Now we sought to better understand the underlying mechanisms. Using lipopolysaccharide (LPS)-induced emergency granulopoiesis as a model, we show that failed expansion was intrinsic to FZD6-deficient HSPCs but also required a FZD6-deficient environment. FZD6-deficient HSPCs became more strongly activated, but their mobilization to peripheral blood was impaired and they were more susceptible to inflammatory cell death, leading to enhanced release of pro-inflammatory cytokines in the marrow. These studies indicate that FZD6 has a protective effect in the bone marrow to prevent an overactive inflammatory response and further suggest that mobilization improves HSPC survival during bone marrow inflammation.

Abstract 29 Unprecedented Experience of Cord Blood Banking in Ukraine During Russian Invasion in 2022

IntroductionOn February 24, 2022, Russia started the unprovoked and unjustified attack on the entire territory of Ukraine. Besides tremendous human losses, the war caused the biggest refugee crisis in Europe since World War II. Approximately 7 million people fled the country; women, some pregnant, and children made 90% of them. Cord blood (CB) banking appeared to be an industry especially sensitive to these unprecedented conditions.ObjectiveThe objective of this study was to analyze the experience of CB banking in Ukraine during the Russia-Ukraine war in 2022.MethodsThematic analysis was performed.ResultsDespite the scarce number of hematopoietic stem cell (SC) transplants for blood malignancies performed there, Ukraine has been a country with a developed regenerative medicine program, with dozens of clinical trials applying perinatal tissues SC. Access to SC therapy was one of the main purposes of medical tourists in Ukraine. The Ukraine’s first CB bank launched in 2003 at the Institute of Cell Therapy LLC and was the main supplier of cell and tissue products to state medical institutions in the country. Thus, the consequences of war, such as disrupted logistics (24,000 km of roads and 300 bridges were destroyed during the first 100 days), ruination of hospitals including maternity clinics (almost 700 health facilities were damaged as of June 9, 2022), lack of human staff (men were mobilized for army and women fled the country), and lack of petroleum, made the successful implementation of contracts on CB storage and SC therapy for both Ukrainian and foreign patients sometimes impossible. The lack of liquid nitrogen experienced during the beginning of the war endangered at least 40,000 CB samples stored at Ukrainian CB banks, as well as biological material at fertility clinics.DiscussionThe unprecedented experience of CB bank operation during the war showed that, for safety reasons, CB banking facilities should be located distantly from strategic objects (eg, railways, airports, and military bases), and should have significant reserve storages of liquid nitrogen, generation reserves, bomb shelter. However, having dedicated personnel was the most important factor allowing for the enterprise functioning in force-majeure events.

Endocytosis of the thrombopoietin receptor Mpl regulates megakaryocyte and erythroid maturation in mice.

Dnm2fl/fl Pf4-Cre (Dnm2Plt–/– ) mice lacking the endocytic GTPase dynamin 2 (DNM2) in platelets and megakaryocytes (MKs) develop hallmarks of myelofibrosis. At the cellular level, the tyrosine kinase JAK2 is constitutively active but decreased in expression in Dnm2Plt–/– platelets. Additionally, Dnm2Plt–/– platelets cannot endocytose the thrombopoietin (TPO) receptor Mpl, leading to elevated circulating TPO levels. Here, we assessed whether the hyperproliferative phenotype of Dnm2Plt–/– mice was due to JAK2 constitutive activation or to elevated circulating TPO levels. In unstimulated Dnm2Plt–/– platelets, STAT3 and, to a lower extent, STAT5 were phosphorylated, but their phosphorylation was slowed and diminished upon TPO stimulation. We further crossed Dnm2Plt–/– mice in the Mpl–/– background to generate Mpl–/–Dnm2Plt–/– mice lacking Mpl ubiquitously and DNM2 in platelets and MKs. Mpl–/– Dnm2Plt–/– platelets had severely reduced JAK2 and STAT3 but normal STAT5 expression. Mpl–/– Dnm2Plt–/– mice had severely reduced bone marrow MK and hematopoietic stem and progenitor cell numbers. Additionally, Mpl–/– Dnm2Plt–/– mice had severe erythroblast (EB) maturation defects, decreased expression of hemoglobin and heme homeostasis genes and increased expression of ribosome biogenesis and protein translation genes in spleen EBs, and developed anemia with grossly elevated plasma erythropoietin (EPO) levels, leading to early fatality by postnatal day 25. Mpl–/– Dnm2Plt+/+ mice had impaired EB development at three weeks of age, which normalized with adulthood. Together, the data shows that DNM2-dependent Mpl-mediated endocytosis in platelets and MKs is required for steady-state hematopoiesis and provides novel insights into a developmentally controlled role for Mpl in normal erythropoiesis, regulating hemoglobin and heme production.

Identification and characterization of in vitro expanded hematopoietic stem cells

Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved > 200‐fold expansion of functional HSCs, but their molecular characterization has not been possible since the majority of cells are non‐HSCs and single cell‐initiated cultures have substantial clone‐to‐clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non‐HSCs in expansion cultures. By directly linking single‐clone functional transplantation data with single‐clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to proliferating fetal HSCs and reveals a gene expression signature, including Esam, Prdm16, Fstl1, and Palld, that can identify functional HSCs from multiple cellular states. This “repopulation signature” (RepopSig) also enriches for HSCs in human datasets. Together, these findings demonstrate the power of integrating functional and molecular datasets to better derive meaningful gene signatures and opens the opportunity for a wide range of functional screening and molecular experiments previously not possible due to limited HSC numbers.

Endothelial-specific Gata3 expression is required for hematopoietic stem cell generation.

SummaryTo generate sufficient numbers of transplantable hematopoietic stem cells (HSCs) in vitro, a detailed understanding of how this process takes place in vivo is essential. The endothelial-to-hematopoietic transition (EHT), which culminates in the production of the first HSCs, is a highly complex process during which key regulators are switched on and off at precise moments, and that is embedded into a myriad of microenvironmental signals from surrounding cells and tissues. We have previously demonstrated an HSC-supportive function for GATA3 within the sympathetic nervous system and the sub-aortic mesenchyme, but show here that it also plays a cell-intrinsic role during the EHT. It is expressed in hemogenic endothelial cells and early HSC precursors, where its expression correlates with a more quiescent state. Importantly, endothelial-specific deletion of Gata3 shows that it is functionally required for these cells to mature into HSCs, placing GATA3 at the core of the EHT regulatory network.

Psoralen suppresses the phosphorylation of amyloid precursor protein (APP) to inhibit myelosuppression

This study aims to explore the effect of Psoralen on myelosuppression, and investigating the mechanism involved in. The mesenchymal stem cells (MSCs) were treated with CTX to construct cell model of myelosuppression, and then with APP knockdown or overexpression transfection. Cell proliferation, cell apoptosis, bone growth factors, and hematopoietic growth factors were identified. The animal model of myelosuppression syndrome was established by intraperitoneal injection of cyclophosphamide (CTX) into C57BL/6 mice, and then with APP knockdown transfection. The effect of Psoralen on myelosuppression mice with APP knockdown was explored, including observin the number of hematopoietic stem cells and bone marrow MSCs, detecting the degree of osteoporosis and the number of osteoclasts. The expression of phosphorylation-amyloid precursor protein (p-APP), bone growth factors, and hematopoietic growth factors were also examined. We found that CTX treatment inhibited cell proliferation, induced cell apoptosis, promoted p-APP/APP, and inhibited the expression of aph-1 homolog A (APH-1α), presenilin enhancer-2 (PEN-2), the receptor of advanced glycation endproducts (RAGE). Psoralen pretreatment effectively promoted cell proliferation, suppressed cell apoptosis, inhibited p-APP/APP and stimulated the expression of APH-1α, PEN-2, RAGE compared with CTX treatment. After APP knockdown, cell proliferation was inhibited, and cell apoptosis was increased. The release of bone growth factors and hematopoietic growth factors was decreased. Psoralen pretreatment could reverse the effect of APP knockdown on MSCs and myelosuppression mice. In conclusion, Psoralen treatment inhibited cell apoptosis and regulated bone growth factors and hematopoietic growth factors in myelosuppression syndrome by suppressing the phosphorylation of APP.

Bioengineered 3D microfibrous-matrix modulates osteopontin release from MSCs and facilitates the expansion of hematopoietic stem cells.

The osteopontin (OPN) released from mesenchymal stem cells (MSCs) undergoing lineage differentiation can negatively influence the expansion of hematopoietic stem cells (HSCs) in coculture systems developed for expanding HSCs. Therefore, minimizing the amount of OPN in the coculture system is important for the successful ex vivo expansion of HSCs. Towardthis goal, a bioengineered three dimensional (3D) microfibrous-matrix that can maintain MSCs in less OPN-releasing conditions has been developed, and its influence on the expansion of HSCs has been studied. The newly developed 3D matrix significantly decreased the release of OPN, depending on the MSC culture conditions used during the priming period before HSC seeding. The culture system with the lowest amount of OPN facilitated a more than 24-fold increase in HSC number in 1 week time period. Interestingly, the viability of expanded cells and the CD34+ pure population of HSCs were found to be the highest in the low OPN-containing system. Therefore, bioengineered microfibrous 3D matrices seeded with MSCs, primed under suitable culture conditions, can be an improved ex vivo expansion system for HSC culture.

Beneficial Modulatory Effects of Treatment With Bone Marrow Lysate on Hematopoietic Stem Cells and Myeloid Cells in Tumor-Bearing Mice.

Introduction: Leukopenia is one of the major side effects of myelosuppressive chemotherapy such as cyclophosphamide (CTX). We and others have used CTX either alone or in combination with G-CSF for the mobilization of hematopoietic stem cells (HSCs). This mobilization can induce expansion of myeloid cells with immunosuppressive phenotype. In this pilot study, we aimed to test whether bone marrow lysate (BML)/CTX, a rich source of growth factors, can lower the expansion of myeloid cells with immunosuppressive phenotypes in tumor-bearing mice without interfering with the anti-tumor effects of CTX or with the mobilization of HSCs. Methods: Female CD1 mice were treated on day 0 with an i.p. injection of Ehrlich ascites carcinoma (EAC). On day 7, the mice were i.p. injected with CTX followed by s.c. injection of G-CSF for 5 consecutive days, single s.c. injection of BML/PBS or BML/CTX or single i.v. injection of BMC/PBS or BMC/CTX. Results: Treatment of EAC-bearing mice with BML/PBS or BML/CTX did not interfere with the anti-tumor effect of CTX. EAC increased the numbers of immature polymorphonuclear cells (iPMN; neutrophils) in both blood and spleen. Treatment of EAC-bearing mice with CTX further increased the numbers of these cells, which were decreased upon treatment with BML/CTX. Treatment with BML/PBS or BML/CTX increased the numbers of stem cells (C.Kit+Sca-1+) in BM; the effect of BML/CTX was higher, but with no significant effect on the numbers of HSCs. Future studies are needed to analyze the molecular components in BM lysate and to determine the underlying mechanisms.

P932: ON-DEMAND PLERIXAFOR WITH CYCLOPHOSPHAMIDE AND G-CSF FOR HEMATOPOIETIC STEM-CELL MOBILIZATION IN MULTIPLE MYELOMA PATIENTS: FINAL RESULTS OF THE MOZOBL06877 STUDY

Background: High-dose melphalan and autologous stem-cell transplant (ASCT) are pillars for the upfront treatment of ASCT-eligible newly diagnosed multiple myeloma (ND)MM patients (pts) or as salvage strategy at relapse. Failure to collect an adequate number of hematopoietic stem cells (HSC) to receive ASCT (<2×106 CD34+ cells/kg) occurs in 5%-15% of MM pts undergoing HSC mobilization with granulocyte colony-stimulating factor (G-CSF) or G-CSF+cyclophosphamide (G-CSF/CY). Aims: We report the final results of the observational MOZOBL06877 study (NCT03406091; partially supported by Sanofi investigation funds) to prospectively assess the performance of HSC mobilization with G-CSF/CY plus on-demand CXCR4 inhibitor plerixafor (PLX) in NDMM pts treated with novel agents. Methods: NDMM pts undergoing HSC mobilization with CY (2-4 g/m2) and G-CSF (5-10 mcg/kg/day) were enrolled and observed up to 30 days after mobilization. According to its label, “on-demand” PLX was administered in pts with <20 CD34+ cells/ul after ≥4 days of G-CSF or in case <1×106 CD34+ cells/kg were collected on the first apheresis day. Pts gave written informed consent. The primary endpoint was the poor mobilizer rate, defined as the rate of pts collecting <2×106 CD34+ cells/kg or requiring PLX. Secondary endpoints were the identification of predictive factors for PLX use and safety during mobilization. Results: 301 NDMM pts were enrolled and analyzed; 72% received induction with bortezomib-thalidomide-dexamethasone, 9% a lenalidomide (Len)-based and 3% a daratumumab (Dara)-based regimen. Overall, 48 pts (16%) were poor mobilizers: 14 (5%) failed to yield ≥2×106/Kg CD34+, while 34 (11%) required rescue with PLX. Among pts yielding <2×106/Kg CD34+ cells, 4 received PLX, while 10 did not. Among pts who successfully collected ≥2×106/Kg CD34+ (n=287, 95%), 34 (12%) required PLX: 23 (68%) due to a CD34+/uL count ≤20 after ≥4 days of G-CSF and 11 (32%) to a collection <1×106/Kg after the first apheresis day. In pts mobilized with PLX, median number of CD34+×106/L cells increased from 17.5 (IQR 10.8-25.6) before PLX to 58.3 (IQR 34.2-100.2) after PLX. Median number of CD34+/Kg collected in pts who did not require PLX was 10.2×106 (IQR 8.3-13.2). In pts who required PLX, a median of 6.5×106 CD34+/Kg (IQR 4.6-9.6) was collected. An adequate HSC yield for ≥2 ASCTs (≥4×106/Kg) was obtained in 96% and 85% of pts not receiving or receiving PLX, respectively. Median number of apheresis days was 1 (IQR 1-2) in pts not requiring PLX and 2 (IQR 1-2) in pts requiring PLX. In a multivariate analysis (Table), factors predicting PLX use were advanced disease stage (R-ISS 3 vs 1-2, OR 5.5, P=0.01), bone marrow (BM) plasma cell (PC) infiltration at diagnosis (PC>60% vs ≤60%, OR 4.2, P<0.001), pre-mobilization absolute neutrophil count (ANC, < vs >2500/mmc, OR 2.8, P=0.01), and the use of Len-based (Yes vs No, OR 3.4, P=0.02) or Dara-based induction regimens (Yes vs No, OR 5.3; P=0.04). Grade 3 infections occurred in 3 pts (1%), with no grade 4-5 reported. Image:Summary/Conclusion: Among NDMM pts treated with novel-agent-based induction and undergoing HSC mobilization with CY/GCSF, “on-demand” PLX in case of low CD34+ cell count or insufficient HSC yield was a safe and effective rescue strategy, reducing mobilization failure from 16% to 5%. Predictive factors for PLX use were advanced disease stage, high BM plasmacytosis, low ANC values before mobilization and the use of Len/Dara during induction. Preemptive PLX in pts with ≥1 risk factors should be investigated to further reduce the risk of mobilization failure.

P1412: UNCOUPLING INK4 PROTEINS FROM CDK4/CDK6 IN HEMATOPOIETIC STEM CELLS

Background: Cell-cycle progression is governed by regulatory proteins including cyclin-dependent kinases (CDKs), cyclins and CDK inhibitors. The INK4 family comprises p16INK4a, p15INK4b, p18INK4c and p19INK4d which are CDK4/6 specific inhibitors. Different tumor types display deletion, mutation, or promoter hypermethylation of the INK4 proteins resulting in enhanced CDK4/6 activity. The INK4a-ARF-INK4b locus encodes for p16INK4a and p15INK4b and for the tumor suppressor protein p14ARF (p19ARF in the mouse) and is one of the most frequently mutated or epigenetically silenced site in human malignancies. CDK4R24C/CDK6R31C double knock-in mice, where R24C and R31C mutations make CDK4/6 fully insensitive to INK4 mediated inhibition, show a shortened survival caused by the onset of a variety of tumors including hematopoietic malignancies. This shows the vital importance of a tight controlled inhibition of CDK4/6 activity in hematopoiesis. Aims: We here explore the consequences of INK4 inhibitory loss on CDK4/6 on the hematopoietic stem cell (HSC) compartment using CDK4R24C/CDK6R31C mice. We propose that INK factors regulate important functions in HSCs by direct binding to CDK4/6. This knowledge is required to understand how to best employ CDK inhibitors in therapeutic settings. Methods: We analyzed the composition of the hematopoietic compartment of CDK4R24C/CDK6R31C double knock-in mice under homeostatic conditions. Long-term self-renewal capacity of CDK4R24C/CDK6R31C bone marrow (BM) cells was assessed by serial bone marrow transplantation (BMT). RNAseq was used to investigate the molecular mechanisms underlying the improved self-renewal of CDK4R24C/CDK6R31C HSCs. Using the stem/progenitor cell line HPCLSK enabled us to perform ChIP qPCR to understand the involvement of CDK4R24C/CDK6R31C for Id1 transcriptional regulation. Results: Upon inhibition of CDK4/6 binding to INK4 proteins, HSC numbers in the BM of CDK4R24C/CDK6R31C mice were reduced. Despite the cell number reduction, CDK4R24C/CDK6R31C HSCs displayed an enhanced self-renewal potential during serial BMT. Transcriptome analysis pointed to the involvement of Id1 in enhanced self-renewal. We indeed found Id1 down-regulated in CDK4R24C/CDK6R31C HSCs at the 3rd round of BMT. CDK6 ChIP experiments confirmed that INK4 binding alters binding of CDK6 to the Id1 promoter, which is significantly reduced in CDK6R31C cells. Summary/Conclusion: Despite reduced qHSCs/MPP1 content under homeostatic conditions, CDK4R24C/CDK6R31C HSCs possess an enhanced self-renewal potential elucidated in serial BMT. We propose that a reduced Id1 expression in qHSCs/MPP1 CDK4R24C/CDK6R31C cells explains this phenotype and underlines improved repopulation ability of CDK4R24C/CDK6R31C BM cells. Our data indicate that INK4 binding to CDK6 dictates its function in regulating transcription.

Genetic Knock-out of TNFR1 and TNFR2 in a JAK2-V617F Polycythemia Vera Mouse Model.

Genetic Knock-out of TNFR1 and TNFR2 in a JAK2-V617F Polycythemia Vera Mouse Model.

Transient Inhibition of the JNK Pathway Promotes Human Hematopoietic Stem Cell Quiescence and Engraftment.

The widespread clinical application of cord blood (CB) for hematopoietic stem cell (HSC) transplantation is limited mainly by the inadequate number of hematopoietic stem and progenitor cells (HSPCs) in single CB units, which results in unsuccessful or delayed engraftment in recipients. The identification of agents to promote CB HSPC engraftment has significant therapeutic value. Here, we found that transient inhibition of the JNK pathway increased the HSC frequency in CB CD34+ cells to 13.46-fold. Mechanistic studies showed that inhibition of the JNK pathway upregulated the expression of quiescence-associated and stemness genes in HSCs, preventing HSCs from entering the cell cycle, increasing glucose uptake and accumulating reactive oxygen species (ROS). Importantly, transient inhibition of the JNK pathway during CB CD34+ cell collection also enhanced long-term HSC (LT-HSC) recovery and engraftment efficiency. Collectively, these findings suggest that transient inhibition of the JNK pathway could promote a quiescent state in HSCs by preventing cell cycle entry and metabolic activation, thus enhancing the HSC number and engraftment potential. Together, these findings improve the understanding of the regulatory mechanisms governing HSC quiescence and stemness and have the potential to improve HSC collection and transplantation.

Defects in Long-Term APC Repopulation Ability of Adult Human Bone Marrow Hematopoietic Stem Cells (HSCs) Compared with Fetal Liver HSCs.

Immunodeficient mice reconstituted with immune systems from patients, or personalized immune (PI) mice, are powerful tools for understanding human disease. Compared with immunodeficient mice transplanted with human fetal thymus tissue and fetal liver-derived CD34+ cells administered i.v. (Hu/Hu mice), PI mice, which are transplanted with human fetal thymus and adult bone marrow (aBM) CD34+ cells, demonstrate reduced levels of human reconstitution. We characterized APC and APC progenitor repopulation in human immune system mice and detected significant reductions in blood, bone marrow (BM), and splenic APC populations in PI compared with Hu/Hu mice. APC progenitors and hematopoietic stem cells (HSCs) were less abundant in aBM CD34+ cells compared with fetal liver-derived CD34+ cell preparations, and this reduction in APC progenitors was reflected in the BM of PI compared with Hu/Hu mice 14-20 wk posttransplant. The number of HSCs increased in PI mice compared with the originally infused BM cells and maintained functional repopulation potential, because BM from some PI mice 28 wk posttransplant generated human myeloid and lymphoid cells in secondary recipients. Moreover, long-term PI mouse BM contained functional T cell progenitors, evidenced by thymopoiesis in thymic organ cultures. Injection of aBM cells directly into the BM cavity, transgenic expression of hematopoietic cytokines, and coinfusion of human BM-derived mesenchymal stem cells synergized to enhance long-term B cell and monocyte levels in PI mice. These improvements allow a sustained time frame of 18-22 wk where APCs and T cells are present and greater flexibility for modeling immune disease pathogenesis and immunotherapies in PI mice.

Cellular and Molecular Mechanisms Involved in Hematopoietic Stem Cell Aging as a Clinical Prospect.

There is a hot topic in stem cell research to investigate the process of hematopoietic stem cell (HSC) aging characterized by decreased self-renewal ability, myeloid-biased differentiation, impaired homing, and other abnormalities related to hematopoietic repair function. It is of crucial importance that HSCs preserve self-renewal and differentiation ability to maintain hematopoiesis under homeostatic states over time. Although HSC numbers increase with age in both mice and humans, this cannot compensate for functional defects of aged HSCs. The underlying mechanisms regarding HSC aging have been studied from various perspectives, but the exact molecular events remain unclear. Several cell-intrinsic and cell-extrinsic factors contribute to HSC aging including DNA damage responses, reactive oxygen species (ROS), altered epigenetic profiling, polarity, metabolic alterations, impaired autophagy, Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, nuclear factor- (NF-) κB pathway, mTOR pathway, transforming growth factor-beta (TGF-β) pathway, and wingless-related integration site (Wnt) pathway. To determine how deficient HSCs develop during aging, we provide an overview of different hallmarks, age-related signaling pathways, and epigenetic modifications in young and aged HSCs. Knowing how such changes occur and progress will help researchers to develop medications and promote the quality of life for the elderly and possibly alleviate age-associated hematopoietic disorders. The present review is aimed at discussing the latest advancements of HSC aging and the role of HSC-intrinsic factors and related events of a bone marrow niche during HSC aging.