IU Indianapolis ScholarWorks :: Browsing by Subject "Hematopoietic stem cells"

Browsing by Subject "Hematopoietic stem cells"

Now showing 1 - 10 of 64

Abstract 16: Insights into Highly Engraftable Hematopoietic Cells from 27-Year Cryopreserved Umbilical Cord Blood
(Oxford University Press, 2023-09-04) Broxmeyer, Hal; Luchsinger, Larry; Weinberg, Rona; Jimenez, Alexandra; Masson Frenet, Emeline; van't Hof, Wouter; Capitano, Maegan; Hillyer, Christopher; Kaplan, Mark; Cooper, Scott; Ropa, James; Microbiology and Immunology, School of Medicine
Introduction: Cord blood banking has consistently outpaced the utilization of cord blood units (CBUs). Thus, the average duration of cryopreservation among banked CBUs will likely continue to increase. It remains unclear how long cryopreserved CBUs remain functional, and it is critical to determine whether duration of cryopreservation should be used as an exclusionary criterion during selection for clinical use or if alternative post-thaw metrics can identify potent cryopreserved CBUs regardless of age. Objectives: Our goal was to determine whether long-term (27-year) cryopreserved CBUs retain viable and functional hematopoietic stem (HSCs) and progenitor cells (HPCs). We further sought to leverage differences in HSC/HPC function (measured by in vivo engraftment) to demonstrate the utility of using omics approaches to identify candidate genes for use as molecular potency markers. Methods: We performed comprehensive ex vivo, in vivo, and molecular analyses on the numbers, viability, and function of three 27-year cryopreserved CBUs using 3-year cryopreserved and fresh CBUs for comparison. Assays included viability staining, immunophenotyping by flow cytometry, primary and secondary colony forming unit (CFU) assays, ex vivo expansion of immunophenotypic HSCs/HPCs/CFUs, limiting dilution transplantations into immune-deficient mice, secondary transplantations, and RNA-sequencing of sorted HSCs and multipotent progenitor cells. Results: Compared to fresh and recently cryopreserved CBU controls, long-term cryopreserved CBUs yield statistically similar numbers of viable immunophenotypic HSCs, multipotent HPCs, and committed myeloid and lymphoid HPCs. They retain highly functional cells, demonstrating similar primary and secondary CFU numbers and expansion capacity compared to controls, as well as robust engraftment, SCID repopulating cell frequency, and secondary engraftment capacity in mouse models of transplantation. Transcriptomic modelling revealed 18 genes, including MALT1 and MAP2K1, and several gene programs, including lineage determination programs and oxidative stress responses, that are strongly enriched in high engrafting HSCs/HPCs. Discussion: CBUs cryopreserved for up to 27 years retain highly functional HSCs/HPCs. Thus, duration of cryopreservation alone is not an ideal exclusionary criteria for selection of CBUs. Preserving older CBUs may help to maintain a large and diverse pool of donors for clinical selection. Further, transcriptomics can identify candidate genes associated with engraftment for elucidation of possible CBU potency markers regardless of the duration of cryopreservation.
Abstract 26: The Role of Oxygen in Cord Blood Hematopoietic Stem and Progenitor Cell Expansion and Engraftment
(Oxford University Press, 2023-09-04) Ropa, James; Gutch, Sarah; Beasley, Lindsay; Van't Hof, Wouter; Kaplan, Mark; Capitan, Maegan; Microbiology and Immunology, School of Medicine
Introduction: Hematopoietic stem (HSC) and progenitor cells (HPCs) are exposed to differing oxygen tensions ranging from <1% to 21% as they reside in/move through different tissues or are harvested for clinical utility. Functional changes in HSCs/HPCs are induced by acute changes in oxygen tension (e.g., a change in percent of cells in cycle). Objectives: We sought to determine if variable oxygen levels affect expansion and/or functional properties of cord blood (CB) HSCs/HPCs ex vivo and in vivo. Methods: Human CB CD34+ cells were grown in expansion culture +/-UM171, an agonist of HSC self-renewal that expands transplantable CB HSCs, in five oxygen tensions: 1%O2, 3%O2, 5%O2, 14%O2, and 21%O2. HSCs/HPCs were enumerated by flow cytometry. Functional HPCs were enumerated by plating in semi solid media for colony forming unit assays (CFU). Cell cycle and reactive oxygen species (ROS) were measured by flow cytometry. Ability of expanded cells to engraft was determined by transplantation in non-lethally irradiated NSG mice. Results: Immunophenotypic HPCs and functional HPC CFUs expanded significantly more after 7 days of growth in higher oxygen tensions (5%O2-21%O2) compared to lower (1%O2-3%O2), while immunophenotypic HSCs expanded best at 5% O2. HSCs/HPCs grown in low oxygen tensions had significantly lower ROS levels, significantly higher percentage of cells in G0, and were slightly but reproducibly smaller/less granular than those grown in high oxygen levels. HSC/HPC numbers were reduced in high oxygen tensions 1-2 days after plating but were better maintained in low, suggesting cells undergo a culture shock/stress after plating that is mitigated by reduced oxygen. In the presence of UM171, HSCs expanded significantly better at higher oxygen levels, but HPCs are better maintained in 5%O2. Ex vivo CD34+ expansions maintained under physiological O2 levels (1-14%O2) demonstrated significantly better/faster neutrophil recovery following transplantation compared to cells expanded at 21%O2 or input. Discussion: HSCs/HPCs proliferate rapidly in high oxygen but have fewer quiescent cells, higher ROS, and are larger and more granular which are all characteristics associated with exhaustion. While high oxygen allows for faster growth, low tensions may mitigate cell stress and allow for prolonged growth (i.e., HSC/HPC expansion) while maintaining functional properties.
ADGRG1 enriches for functional human hematopoietic stem cells following ex vivo expansion-induced mitochondrial oxidative stress
(The American Society for Clinical Investigation, 2021) Chen, Yandan; Fang, Shuyi; Ding, Qingwei; Jiang, Rongzhen; He, Jiefeng; Wang, Qin; Jin, Yuting; Huang, Xinxin; Liu, Sheng; Capitano, Maegan L.; Trinh, Thao; Teng, Yincheng; Meng, Qingyou; Wan, Jun; Broxmeyer, Hal E.; Guo, Bin; BioHealth Informatics, School of Informatics and Computing
The heterogeneity of human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) under stress conditions such as ex vivo expansion is poorly understood. Here, we report that the frequencies of SCID-repopulating cells were greatly decreased in cord blood (CB) CD34+ HSCs and HPCs upon ex vivo culturing. Transcriptomic analysis and metabolic profiling demonstrated that mitochondrial oxidative stress of human CB HSCs and HPCs notably increased, along with loss of stemness. Limiting dilution analysis revealed that functional human HSCs were enriched in cell populations with low levels of mitochondrial ROS (mitoROS) during ex vivo culturing. Using single-cell RNA-Seq analysis of the mitoROS low cell population, we demonstrated that functional HSCs were substantially enriched in the adhesion GPCR G1-positive (ADGRG1+) population of CD34+CD133+ CB cells upon ex vivo expansion stress. Gene set enrichment analysis revealed that HSC signature genes including MSI2 and MLLT3 were enriched in CD34+CD133+ADGRG1+ CB HSCs. Our study reveals that ADGRG1 enriches for functional human HSCs under oxidative stress during ex vivo culturing, which can be a reliable target for drug screening of agonists of HSC expansion.
Age and Sex Divergence in Hematopoietic Radiosensitivity in Aged Mouse Models of the Hematopoietic Acute Radiation Syndrome
(BioOne, 2022) Patterson, Andrea M.; Vemula, Sasidhar; Plett, P. Artur; Sampson, Carol H.; Chua, Hui Lin; Fisher, Alexa; Wu, Tong; Sellamuthu, Rajendran; Feng, Hailin; Katz, Barry P.; DesRosiers, Colleen M.; Pelus, Louis M.; Cox, George N.; MacVittie, Thomas J.; Orschell, Christie M.; Medicine, School of Medicine
The hematopoietic system is highly sensitive to stress from both aging and radiation exposure, and the hematopoietic acute radiation syndrome (H-ARS) should be modeled in the geriatric context separately from young for development of age-appropriate medical countermeasures (MCMs). Here we developed aging murine H-ARS models, defining radiation dose response relationships (DRRs) in 12-month-old middle-aged and 24-month-old geriatric male and female C57BL/6J mice, and characterized diverse factors affecting geriatric MCM testing. Groups of approximately 20 mice were exposed to ∼10 different doses of radiation to establish radiation DRRs for estimation of the LD50/30. Radioresistance increased with age and diverged dramatically between sexes. The LD50/30 in young adult mice averaged 853 cGy and was similar between sexes, but increased in middle age to 1,005 cGy in males and 920 cGy in females, with further sex divergence in geriatric mice to 1,008 cGy in males but 842 cGy in females. Correspondingly, neutrophils, platelets, and functional hematopoietic progenitor cells were all increased with age and rebounded faster after irradiation. These effects were higher in aged males, and neutrophil dysfunction was observed in aged females. Upstream of blood production, hematopoietic stem cell (HSC) markers associated with age and myeloid bias (CD61 and CD150) were higher in geriatric males vs. females, and sex-divergent gene signatures were found in HSCs relating to cholesterol metabolism, interferon signaling, and GIMAP family members. Fluid intake per gram body weight decreased with age in males, and decreased after irradiation in all mice. Geriatric mice of substrain C57BL/6JN sourced from the National Institute on Aging were significantly more radiosensitive than C57BL/6J mice from Jackson Labs aged at our institution, indicating mouse source and substrain should be considered in geriatric radiation studies. This work highlights the importance of sex, vendor, and other considerations in studies relating to hematopoiesis and aging, identifies novel sex-specific functional and molecular changes in aging hematopoietic cells at steady state and after irradiation, and presents well-characterized aging mouse models poised for MCM efficacy testing for treatment of acute radiation effects in the elderly.
Age-related decline in LEPR+ hematopoietic stem cell function
(Springer Nature, 2023) Trinh, Thao; Ropa, James; Cooper, Scott; Aljoufi, Arafat; Sinn, Anthony; Capitano, Maegan; Broxmeyer, Hal E.; Kaplan, Mark H.; Microbiology and Immunology, School of Medicine
Aging-Related Reduced Expression of CXCR4 on Bone Marrow Mesenchymal Stromal Cells Contributes to Hematopoietic Stem and Progenitor Cell Defects
(SpringerLink, 2020-08) Singh, Pratibha; Kacena, Melissa A.; Orschell, Christie M.; Pelus, Louis M.; Microbiology and Immunology, School of Medicine
Aging impairs the regenerative potential of hematopoietic stem cells (HSC) and skews differentiation towards the myeloid lineage. The bone marrow (BM) microenvironment has recently been suggested to influence HSC aging, however the mechanisms whereby BM stromal cells mediate this effect is unknown. Here we show that aging-associated decreased expression of CXCR4 expression on BM mesenchymal stem cells (MSC) plays a crucial role in the development of the hematopoietic stem and progenitor cells (HSPC) aging phenotype. The BM MSC from old mice was sufficient to drive a premature aging phenotype of young HSPC when cultured together ex vivo. The impaired ability of old MSC to support HSPC function is associated with reduced expression of CXCR4 on BM MSC of old mice. Deletion of the CXCR4 gene in young MSC accelerates an aging phenotype in these cells characterized by increased production of reactive oxygen species (ROS), DNA damage, senescence, and reduced proliferation. Culture of HSPC from young mice with CXCR4 deficient MSC also from young mice led to a premature aging phenotype in the young HSPC, as evidenced by reduced hematopoietic regeneration and enhanced myeloid differentiation. Mechanistically, CXCR4 signaling prevents BM MSC dysfunction by suppressing oxidative stress, as treatment of old or CXCR4 deficient MSC with N-acetyl-L-cysteine (NAC), improved their niche supporting activity, and attenuated the HSPC aging phenotype. Our studies suggest that age-associated reduction in CXCR4 expression on BM MSC impairs hematopoietic niche activity with increased ROS production, driving an HSC aging phenotype. Thus, modulation of the SDF-1/CXCR4 axis in MSC may lead to novel interventions to alleviate the age-associated decline in immune/hematopoietic function.
Alkynyl nicotinamides show antileukemic activity in drug-resistant acute myeloid leukemia
(The American Society for Clinical Investigation, 2024-06-17) Ramdas, Baskar; Dayal, Neetu; Pandey, Ruchi; Larocque, Elizabeth; Kanumuri, Rahul; Pasupuleti, Santhosh Kumar; Liu, Sheng; Kanellopoulou, Chrysi; Chu, Elizabeth Fei Yin; Mohallem, Rodrigo; Virani, Saniya; Chopra, Gaurav; Aryal, Uma K.; Lapidus, Rena; Wan, Jun; Emadi, Ashkan; Haneline, Laura S.; Holtsberg, Frederick W.; Aman, M. Javad; Sintim, Herman O.; Kapur, Reuben; Pediatrics, School of Medicine
Activating mutations of FLT3 contribute to deregulated hematopoietic stem and progenitor cell (HSC/Ps) growth and survival in patients with acute myeloid leukemia (AML), leading to poor overall survival. AML patients treated with investigational drugs targeting mutant FLT3, including Quizartinib and Crenolanib, develop resistance to these drugs. Development of resistance is largely due to acquisition of cooccurring mutations and activation of additional survival pathways, as well as emergence of additional FLT3 mutations. Despite the high prevalence of FLT3 mutations and their clinical significance in AML, there are few targeted therapeutic options available. We have identified 2 novel nicotinamide-based FLT3 inhibitors (HSN608 and HSN748) that target FLT3 mutations at subnanomolar concentrations and are potently effective against drug-resistant secondary mutations of FLT3. These compounds show antileukemic activity against FLT3ITD in drug-resistant AML, relapsed/refractory AML, and in AML bearing a combination of epigenetic mutations of TET2 along with FLT3ITD. We demonstrate that HSN748 outperformed the FDA-approved FLT3 inhibitor Gilteritinib in terms of inhibitory activity against FLT3ITD in vivo.
Antagonism of PPARγ signaling expands human hematopoietic stem and progenitor cells by enhancing glycolysis
(Nature Publishing group, 2018-03) Guo, Bin; Huang, Xinxin; Lee, Man Ryul; Lee, Sang A; Broxmeyer, Hal E.; Microbiology and Immunology, School of Medicine
Hematopoietic stem cells (HSCs) quiescently reside in bone marrow niches and have the capacity to self-renew or differentiate to form all blood cells throughout the lifespan of an animal–. Allogeneic HSC transplantation is a life-saving treatment for malignant and non-malignant disorders,. HSCs isolated from umbilical cord blood (CB) are used for hematopoietic cell transplantation (HCT)–, but due to limited numbers of HSCs in single units of umbilical CB, a number of methods have been proposed for ex vivo expansion of human HSCs,,. We show here that antagonism of the nuclear hormone receptor PPARγ promotes ex vivo expansion of phenotypically and functionally-defined subsets of human CB HSCs and hematopoietic progenitor cells (HSPCs). PPARγ antagonism in CB HSPCs strongly downregulated expression of several differentiation associated genes, as well as fructose 1, 6-bisphosphatase (FBP1), a negative regulator of glycolysis, and enhanced glycolysis without compromising mitochondrial metabolism. The expansion of CB HSPCs by PPARγ antagonism was completely suppressed by removal of glucose or inhibition of glycolysis. Moreover, knockdown of FBP1 expression promoted glycolysis and ex vivo expansion of long-term repopulating CB HSPCs, whereas overexpression of FBP1 suppressed the expansion of CB HSPCs induced by PPARγ antagonism. Our study suggests the possibility for a new and simple means for metabolic reprogramming of CB HSPCs to improve the efficacy of HCT.
CD166 Engagement Augments Mouse and Human Hematopoietic Progenitor Function via Activation of Stemness and Cell Cycle Pathways
(Oxford University Press, 2019) Zhang, Jing; Ghosh, Joydeep; Mohamad, Safa F.; Zhang, Chi; Huang, Xinxin; Capitano, Maegan L.; Gunawan, Andrea M.; Cooper, Scott; Guo, Bin; Cai, Qingchun; Broxmeyer, Hal E.; Srour, Edward F.; Microbiology and Immunology, School of Medicine
Hematopoietic stem (HSC) and progenitor (HPC) cells are regulated by interacting signals and cellular and noncellular elements of the hematopoietic niche. We previously showed that CD166 is a functional marker of murine and human HSC and of cellular components of the murine niche. Selection of murine CD166+ engrafting HSC enriched for marrow repopulating cells. Here, we demonstrate that CD166-CD166 homophilic interactions enhance generation of murine and human HPC in vitro and augment hematopoietic function of these cells. Interactions between cultured CD166+ Lineage- Sca-1+ c-Kit+ (LSK) cells and CD166+ osteoblasts (OBs) significantly enhanced the expansion of colony-forming units (CFUs). Interactions between CD166+ LSK cells and immobilized CD166 protein generated more CFU in short-term cultures than between these cells and bovine serum albumin (BSA) or in cultures initiated with CD166- LSK cells. Similar results were obtained when LSK cells from wildtype (WT) or CD166 knockout (KO) (CD166-/- ) mice were used with immobilized CD166. Human cord blood CD34+ cells expressing CD166 produced significantly higher numbers of CFUs following interaction with immobilized CD166 than their CD166- counterparts. These data demonstrate the positive effects of CD166 homophilic interactions involving CD166 on the surface of murine and human HPCs. Single-cell RNA-seq analysis of CD150+ CD48- (signaling lymphocyte activation molecule (SLAM)) LSK cells from WT and CD166-/- mice incubated with immobilized CD166 protein revealed that engagement of CD166 on these cells activates cytokine, growth factor and hormone signaling, epigenetic pathways, and other genes implicated in maintenance of stem cell pluripotency-related and mitochondria-related signaling pathways. These studies provide tangible evidence implicating CD166 engagement in the maintenance of stem/progenitor cell function.
CD166 modulates disease progression and osteolytic disease in multiple myeloma
(2016-03-16) Xu, Linlin; Xu, Linlin
Multiple myeloma (MM) is an incurable malignancy characterized by the proliferation of neoplastic plasma cells in the bone marrow (BM) and by multiple osteolytic lesions throughout the skeleton. We previously reported that CD166 is a functional molecule on normal hematopoietic stem cells (HSC) that plays a critical role in HSC homing and engraftment, suggesting that CD166 is involved in HSC trafficking and lodgment. CD166, a member of the immunoglobulin superfamily capable of mediating homophilic interactions, has been shown to enhance metastasis and invasion in several tumors. However, whether CD166 is involved in MM and plays a role in MM progression has not been addressed. We demonstrated that a fraction of all human MM cell lines tested and MM patients’ BM CD138+ cells express CD166. Additionally, CD166+ cells preferentially home to the BM of NSG mice. Knocking-down (KD) CD166 expression on MM cells with shRNA reduced their homing to the BM. Furthermore, in a long-term xenograft model, NSG mice inoculated with CD166KD cells showed delayed disease progression and prolonged survival compared to mice receiving mock transduced cells. To examine the potential role of CD166 in osteolytic lesions, we first used a novel Ex Vivo Organ Culture Assay (EVOCA) which creates an in vitro 3D system for the interaction of MM cells with the bone microenvironment. EVOCA data from MM cells lines as well as from primary MM patients’ CD138+ BM cells demonstrated that bone osteolytic resorption was significantly reduced when CD166 was absent on MM cells or calvarial cells. We then confirmed our ex vivo findings with intra-tibial inoculation of MM cells in vivo. Mice inoculated with CD166KD cells had significantly less osteolytic lesions. Further analysis demonstrated that CD166 expression on MM cells alters bone remodeling by inhibiting RUNX2 gene expression in osteoblast precursors and increasing RANKL to OPG ratio in osteoclast precursors. We also identified that CD166 is indispensable for osteoclastogenesis via the activation of TRAF6-dependent signaling pathways. These results suggest that CD166 directs MM cell homing to the BM and promotes MM disease progression and osteolytic disease. CD166 may serve as a therapeutic target in the treatment of MM.

Browsing by Subject "Hematopoietic stem cells"

Results Per Page

Sort Options