Characteristics Of Hematopoietic Stem Cells Research Articles

Advances in hematopoietic stem cells ex vivo expansion associated with bone marrow niche.

Hematopoietic stem cells (HSCs) are an ideal source for the treatment of many hematological diseases and malignancies, as well as diseases of other systems, because of their two important features, self-renewal and multipotential differentiation, which have the ability to rebuild the blood system and immune system of the body. However, so far, the insufficient number of available HSCs, whether from bone marrow (BM), mobilized peripheral blood or umbilical cord blood, is still the main restricting factor for the clinical application. Therefore, strategies to expand HSCs numbers and maintain HSCs functions through ex vivo culture are urgently required. In this review, we outline the basic biology characteristics of HSCs, and focus on the regulatory factors in BM niche affecting the functions of HSCs. Then, we introduce several representative strategies used for HSCs from these three sources ex vivo expansion associated with BM niche. These findings have deepened our understanding of the mechanisms by which HSCs balance self-renewal and differentiation and provided a theoretical basis for the efficient clinical HSCs expansion.

Decline in Cardiolipin in Hematopoietic Stem Cell during Aging Alters Their Regenerative Potential

Hematopoietic Stem Cells (HSCs) are known for their regenerative potential. This property allowed the use of HSC in bone marrow transplantation to treat hematological disorders such as leukemia. However, aging influences these characteristics of HSCs. The major hallmarks of aging are limited self-renewal and reconstitution capacity resulting in HSC exhaustion in aged individuals. Mitochondrial metabolism and activity are active drivers of HSC fate decisions and data from our lab shows that mitochondria in aged HSCs have increased sphericity with a polarized mitochondrial network and a lower mitochondrial membrane potential. An important aspect of mitochondrial functions is the lipid composition of their membranes. A lipid trafficking assay showed an atypical pattern of lipid incorporation by mitochondria in aged HSCs suggesting that mitochondrial lipids become abnormal upon aging. The overall content of Cardiolipin (CL) which is a signature mitochondrial lipid, found exclusively in the inner mitochondrial membrane was also reduced during aging. This was done with flow cytometry using a CL marker NAO (Non-Acridine Orange). Tafazzin encoded by the gene TAZ is crucial for remodeling CL into its mature form which is required for maintaining mitochondrial functions. Mutations in the TAZ gene have been known to cause a reduction in the synthesis of mature CL which is often associated with HSCs functional defect. Western Blot analysis showed a considerable decrease in Taz protein expression in aged stem and progenitor cells (HSPC) compared to young HSPC. To understand the effect of reduced Taz expression on HSC functions, we used a doxycycline inducible, sh-RNA mediated TAZ knock-down (KD) mouse model. Bone marrow analysis indicates that the frequency of HSC and multipotent progenitors decreases significantly in 6-month-old TAZ KD mice compared to wild type (WT) mice. Our data shows that following 5FU induction TAZ KD mice have a significant reduction in the multipotent progenitor and HSC population on day 10 and day 21 post 5FU, indicating TAZ KD decrease the regenerative potential of HSCs under stress conditions. Further pointing towards the significance of cardiolipin in HSC regeneration. TAZ KD mice also have abnormal peripheral blood recovery post 5FU mainly affecting the platelets. We next examined the repopulation potential of HSCs isolated from TAZ KD mice using serial competitive transplantation assays. While primary transplanted mice of TAZ KD HSCs exhibited similar donor-cell chimerism compared to mice transplanted with WT HSCs, about 50-60%, secondary recipient of TAZ KD cells had significantly lower donor-cell chimerism than secondary recipients of WT cells. Further, donor-derived HSC frequency in the bone marrow of recipients of TAZ KD HSCs 24 weeks following engraftment was also significantly lower than recipients of WT HSCs. Hence, Taz-deficiency causes a defect in HSC self-renewal under regenerative conditions. We next evaluated whether restoring cardiolipin levels in mid-aged HSCs could rescue their functions. For this, mid-aged HSC were incubated for 3 days under conditions that maintain HSC functions in vitro with Alcar (Acetyl-L-Carnitine), which promotes CL production. In lipid incorporation assay, Alcar supplementation restored mitochondrial membrane potential, mitochondrial organization and lipid incorporation of mid-aged HSCs. In competitive transplant assay, while mid-aged WT HSC treated with vehicle gave rise to a myeloid-bias graft, Alcar supplemented mid-aged WT HSC gave rise to a more balanced graft with increased ratio lymphoid to myeloid cells in the peripheral blood 16 weeks following transplantation, albeit the rescue was partial. This study indicates that HSC mitochondrial membrane lipids become abnormal with aging which causes a decline in HSC function and highlights the critical role of mitochondrial cardiolipin in HSC functions. It implies that metabolite supplementation is a viable option for restoring age-related HSC functional defects.

The AP-1/ETS Transcription Factor Network Regulates Stem Cell Quiescence and Chemotherapy Resistance in Acute Myeloid Leukemia

Despite of the continuous improvements in the treatment for acute myeloid leukemia (AML), therapy resistance and disease relapse have been persistent and attributed to leukemia stem cells (LSCs). Recent single cell RNA sequencing (Galen et al, Bernstein 2019) and deconvoluted bulk RNA sequencing analysis (Zeng et al, Dick 2022) show hierarchical cellular organization in individual AML patient specimens, with LSCs, and quiescent LSCs in particular, at its apex. However, the mechanisms controlling LSCs and their quiescence remain elusive, hindering the development of effective targeted therapies. Here, using human patient AML specimens, we show that activator protein 1 (AP-1) and E26 transformation-specific (ETS) family transcription factors (TFs) coordinately regulate LSC quiescence and disease persistence after chemotherapy. A chemical label tracing technique using carboxyfluorescein succinimidyl ester (CFSE) has emerged as a powerful tool to identify quiescent LSCs, and using this technique, we and others have demonstrated that CFSE-label retaining quiescent LSCs isolated from patient leukemia cells exhibit potent leukemia initiating property, resistance to conventional chemotherapy, and epigenetic plasticity (Takao et al, Kentsis 2018 ASH, Ebinger et al, Jeremias 2016 and 2020). Here, we combined this method with functional genomic approaches to elucidate the mechanisms controlling quiescent LSCs and identify novel therapeutic targets to overcome chemotherapy resistance. First, to identify the candidate genes for LSC quiescence regulators, we performed gene expression and chromatin accessibility profiling of label-retaining quiescent LSCs isolated from human patient leukemia cell-engrafted NSG mice in diverse subtypes of AMLs using mRNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). We identified that several transcription factors important for normal hematopoiesis, which included AP-1 TFs, such as JUN and JUNB, and ETS TFs, such as ETS1 and FLI1, were differentially expressed in quiescent LSCs. Chromatin accessibility profiling further revealed the predominant occupancy of AP-1 and ETS TF family binding motifs in differentially accessible chromatin loci in quiescent LSCs. Importantly, gene expression signatures detected in quiescent LSCs showed significant similarity to gene expression profiles of residual leukemia cells observed in chemotherapy-treated AML patients (GSE116256, Galen et al. 2019), supporting the clinical relevance of our models. Next, to develop a functional screen system to identify the regulators of quiescent LSCs in human patient AML-bearing mice, we designed a lentiviral vector encoding mCherry fluorescent protein and doxycycline-inducible cDNAs marked by specific barcode sequences. cDNA library screening using this lentiviral vector identified several TFs as regulators for AML LSC quiescence, which included JUN as a positive regulator, and ETS1 and FLI1 as negative regulators (Figure 1). Finally, given the evidence of transcriptional activity of JUN to regulate LSC quiescence, we hypothesized that JUN activity may be also associated with chemotherapy resistance and disease persistence in patients. To investigate this, we analyzed JUN expression in individual AML cells in diverse patients before and after chemotherapy treatment (GSE116256, Galen et al. 2019). We found JUN to be highly expressed in persistent AML cells upon induction chemotherapy, especially in leukemia cells with features of hematopoietic stem cells (HSC-like), while ETS1 and FLI1 expression was not (Figure 2). These results argue that JUN activity may be required for chemotherapy resistance, LSC persistence, and ultimate AML relapse. In all, human AML LSC quiescence and therapy response can be dynamically regulated by a distinct transcription factor network. This work defines molecular mechanisms controlling AML LSC quiescence. We anticipate that these findings will lead to the elucidation of essential properties of LSC quiescence and the design of therapeutic strategies for their clinical identification and control. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Application of cells of cord blood and umbilical cord: achievements, challenges and perspectives

This review focuses on the biological aspects of the use of cord blood as a valuable source of cells. The distinctive features of hematopoietic stem cells, cells of the immune system, mesenchymal stem cells are described. The analysis of the results of clinical research and development of therapeutic approaches using cord blood cells and umbilical cord for the treatment of various diseases has been carried out. Currently, the target area of cord blood research is hematopoietic stem cell transplantation, as well as cellular immunotherapy of tumor diseases, treatment of neurological diseases and regenerative medicine.

Research on mechanism of tanshinone a in regulating biological characteristics of hematopoietic stem cell in liver cirrhosis through targeting of miR-9a-5p

This study assessed the mechanism of tanshinone A in regulating biological characteristics of Hematopoietic Stem Cell (HSC) in liver cirrhosis through targeting of miR-9a-5p. HSC cells were divided into negative control group and stimulated miR-9a-5p inhibitor group. Transfection was performed according to specification of the kit. Expression of miR-9a-5p was assessed with Real-time polymerase chain reaction (PCR). Cell proliferation was tested with flow cytometry (FCM), and α-smooth muscle actin (SMA) and Type I collagen expressions were detected with Western Blot assay. Caspase-3 activity was tested with spectrophotometry, while variation of inflammatory factor was detected with enzyme-linked immunosorbent assay (ELISA). There was higher miR-9a-5p level in HSC induced by Chemokine (C-C motif) ligands 4 (CCL-4). Biological characteristics of HSC induced by CCL-4 was restrained by down-regulation of miR-9a-5p, and presentation quantity of α-SMA and Type I collagen was reduced. So, occurrence of inflammation and migration of HSC could be restrained. The presentation quantity of Type I collagen was reduced with tanshinone A, and expression of miR-9a-5p was reduced. HSC characteristics in liver cirrhosis were affected by tanshinone A probably through regulating miR-9a-5p. It could provide a brand-new selection for treatment on liver cirrhosis.

Mutant TP53 prevents Telomere Shortening in Acute Myeloid Leukemia

BackgroundHigh telomerase activity represents a critical feature of hematopoietic stem cells. Excessive shortening of telomere length (TL) due to replicative stress may be - in analogy to many solid tumors - a hallmark of myeloid neoplasia (MN). Also, telomeric footprints in leukemic genomes may vary between various subtypes corresponding to the differentiation arrest at various stages of hematopoietic ontogeny or specific molecular defects. Critical TL shortening has been associated with genomic instability and accelerated acquisition of genomic lesions leading to a more aggressive phenotype. These processes have not been systematically studied in MN, especially AML.AimBy taking advantage of next-generation sequencing to assay both molecular features and TL within large cohorts of patients, we tested the hypothesis that TL shortening is excessive in highly proliferative MN, but that distinct invariant differences characterize genetic subtypes.MethodsOur cohort included AML (N=734), MDS (N=701), healthy controls (HC) (N=11) and PNH (N=102) serving as clonal non-malignant controls. All patients were diagnosed according to WHO standards before being subjected to transcriptome (WTS) and genome (WGS, 100x) sequencing. To retrieve TL characteristics and telomere repeat heterogeneity from WGS data, we used TelomereHunter (TH). In parallel, we performed C-Circle assays. Patients were annotated for clinical features and analyzed for genetic/transcriptomic patterns.ResultsFor a subset of patients for whom corresponding benign lymphocyte DNA was available a significant TL shortening in blasts vs control lymphocytes (A; P=.0023) was detected. While age correlation was established in controls, despite a trend, in MN age did not significantly affect TL (B) and thus subsequent comparisons were not adjusted for age. Next, we studied a cohort of patients with AML, MDS, PNH and HC and found that TL shortening was an overarching finding in AML, MDS and PNH as compared to HC (C). Since no matched DNA was available as reference, we examined the distribution of TL across different age cohorts, AML patients divided according to age cohorts harbored TL in a similar range (D, P=.057). Classic morphologic (E) or cytogenetic subtypes AML exhibited no difference. Similarly, no differences were found between high and low risk MDS patients (not shown). The variability of TL ranges suggested that there may be molecular factors which affect individual TL. When we compared TL grouped according to frequent mutations, only TP53 mutations were associated with longer TL (F, P<.0001). A significant positive correlation (G, P=.021) between TL and TP53 clonal burden was found; samples with the longest vs shortest TL showed significantly higher TP53 VAF (H, P=.0229). In analogy, the presence of multiple TP53 mutations (putative biallelic inactivation) showed longer TL than single hits but no association was found between the nature of mutations and TL (I, J, K). Availability of WTS data allowed us to assess the telomerase activity using the EXTEND score (ES) which has been shown to assess telomerase activity. Indeed, the ES was correlated with TL (L) and TP53 mutant status was associated with a higher ES compared to WT samples (M, P<.0001). Similarly, because of the compensatory upregulation of TP53 in mutant cases, we have also found that TP53 mRNA levels correlated with ES (N P<.0001). Another explanation of TL increase could be the occurrence of alternative lengthening (ALT). TH software allows for estimation of the abundance of specific telomeric repeats. Singleton analysis showed that increase in telomere repeats variants (TTTGGG, O, P=.003) was related to mutations in TP53 arguing against the involvement of ALT. The final confirmation that TL extension was not due to ALT was provided by C-Circle assays. When C-Circle assays were performed for samples with a high/low TL and mutant/WT TP53, none of the subgroups was identified as ALT + (P).ConclusionWe stipulate that TL measurements using NGS will be helpful to investigate pathophysiological features associated with TL shortening. Availability of therapies targeting the telomere machinery (Imetelstat) may offer an opportunity for personalized therapy beyond MPN, its current indication. It remains to be tested whether long TL associated with TP53 mutations can serve as marker of sensitivity or resistance to these agents. [Display omitted] DisclosuresHaferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Regeneron: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Alexion: Consultancy; Novartis: Consultancy.

Understanding the "SMART" features of hematopoietic stem cells and beyond.

Since the huge success of bone marrow transplantation technology in clinical practice, hematopoietic stem cells (HSCs) have become the gold standard for defining the properties of adult stem cells (ASCs). Here, we describe the “self-renewal, multi-lineage differentiation, apoptosis, rest, and trafficking” or “SMART” model, which has been developed based on data derived from studies of HSCs as the most well-characterized stem cell type. Given the potential therapeutic applications of ASCs, we delineate the key characteristics of HSCs using this model and speculate on the physiological relevance of stem cells identified in other tissues. Great strides are being made in understanding the biology of ASCs, and efforts are now underway to develop safe and effective ASC-based therapies in this emerging area.

The Therapeutic Potential of Hematopoietic Stem Cells in Bone Regeneration.

The repair process of bone fractures is a complex biological mechanism requiring the recruitment and in situ functionality of stem/stromal cells from the bone marrow (BM). BM mesenchymal stem/stromal cells have been widely explored in multiple bone tissue engineering applications, whereas the use of hematopoietic stem cells (HSCs) has been poorly investigated in this context. A reasonable explanation is the fact that the role of HSCs and their combined effect with other elements of the hematopoietic niches in the bone-healing process is still elusive. Therefore, in this review we intend to highlight the influence of HSCs in the bone repair process, mainly through the promotion of osteogenesis and angiogenesis at the bone injury site. For that, we briefly describe the main biological characteristics of HSCs, as well as their hematopoietic niches, while reviewing the biomimetic engineered BM niche models. Moreover, we also highlighted the role of HSCs in translational in vivo transplantation or implantation as promoters of bone tissue repair. Impact statement The ability of bone to natural self-heal depends on the size and stabilization level of the tissue fracture, and it is impaired in several pathophysiological conditions. Considering that the available treatment options have demonstrated limited regenerative performance, the hematopoietic stem cells (HSCs) co-cultured in different tissue engineering strategies have emerged as a powerful tool to promote effective bone regeneration and healing. Here, we reviewed the most important biomimetic bone-marrow hematopoietic niches and showed the regenerative potential of these cells, both in vitro and in translational in vivo transplantation/implantation approaches. This knowledge encourages the development of new HSC-related bone regenerative therapies.

Mitochondrial calcium homeostasis in hematopoietic stem cell: Molecular regulation of quiescence, function, and differentiation.

Hematopoiesis is based on the existence of hematopoietic stem cells (HSC) with the capacity to self-proliferate and self-renew or to differentiate into specialized cells. The hematopoietic niche is the essential microenvironment where stem cells reside and integrate various stimuli to determine their fate. Recent studies have identified niche containing high level of calcium (Ca2+) suggesting that HSCs are sensitive to Ca2+. This is a highly versatile and ubiquitous second messenger that regulates a wide variety of cellular functions. Advanced methods for measuring its concentrations, genetic experiments, cell fate tracing data, single-cell imaging, and transcriptomics studies provide information into its specific roles to integrate signaling into an array of mechanisms that determine HSC identity, lineage potential, maintenance, and self-renewal. Accumulating and contrasting evidence, are revealing Ca2+ as a previously unacknowledged feature of HSC, involved in functional maintenance, by regulating multiple actors including transcription and epigenetic factors, Ca2+-dependent kinases and mitochondrial physiology. Mitochondria are significant participants in HSC functions and their responsiveness to cellular demands is controlled to a significant extent via Ca2+ signals. Recent reports indicate that mitochondrial Ca2+ uptake also controls HSC fate. These observations reveal a physiological feature of hematopoietic stem cells that can be harnessed to improve HSC-related disease. In this review, we discuss the current knowledge Ca2+ in hematopoietic stem cell focusing on its potential involvement in proliferation, self-renewal and maintenance of HSC and discuss future research directions.

Cleavable, Covalently Linked, Affinity Coupling Immune Magnetic Nanoparticles for Specifically Depleting T Cells

Hematopoietic stem cell (HSC) transplantation has become an accepted therapeutic modality for patients with hematopoietic disorders. However, transplant recipients are at high risk of a severe complication known as graft versus host disease (GvHD). To reduce the risk of GvHD, depletion of T cells in donor HSCs prior to transplantation is of vital importance. Magnetic-activated cell sorting (MACS) using immuno-magnetic nanoparticles to selectively deplete T cells presents a potential solution for HSC transplantation. In this study, we presented a complete protocol for the synthesis of immuno-magnetic nanoparticles and characterised their performance for the depletion of Jurkat T cells from cell culture and from a mixture of T cells and HSCs. First, the recombinant protein A/G, an Fc-specific antibody, and magnetic nanoparticles were covalently linked by amine groups on the surface of magnetic nanoparticles and the protein using 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP). Then, the anti-Jurkat T antibodies were bound to protein A/G on magnetic nanoparticles via regio-oriented affinity binding. Approximately 85 μg of protein A/G and 25 μg of antibody were bound to 1 mg of magnetic beads. The immuno-magnetic nanoparticles were capable of depleting up to 73% of Jurkat T cells from culture medium and 72% of Jurkat T cells from the mixture. In addition, we showed that our depletion protocol was able to preserve the proliferation and differentiation characteristics of HSCs. In conclusion, our immuno-magnetic nanoparticles provide a potential solution for the depletion of T-cells prior to HSCs transplantation.

BMP signaling is required for postnatal murine hematopoietic stem cell self-renewal.

Life-long production of blood from hematopoietic stem cells (HSC) is a process of strict modulation. Intrinsic and extrinsic signals govern fate options like self-renewal – a cardinal feature of HSC. Bone morphogenetic proteins (BMP) have an established role in embryonic hematopoiesis, but less is known about its functions in adulthood. Previously, SMAD-mediated BMP signaling has been proven dispensable for HSC. However, the BMP type-II receptor (BMPR-II) is highly expressed in HSC, leaving the possibility that BMP function via alternative pathways. Here, we establish that BMP signaling is required for selfrenewal of adult HSC. Through conditional knockout we show that BMPR-II deficient HSC have impaired self-renewal and regenerative capacity. BMPR-II deficient cells have reduced p38 activation, implying that non-SMAD pathways operate downstream of BMP in HSC. Indeed, a majority of primitive hematopoietic cells do not engage in SMADmediated responses downstream of BMP in vivo. Furthermore, deficiency of BMPR-II results in increased expression of TJP1, a known regulator of self-renewal in other stem cells, and knockdown of TJP1 in primitive hematopoietic cells partly rescues the BMPR-II null phenotype. This suggests TJP1 may be a universal stem cell regulator. In conclusion, BMP signaling, in part mediated through TJP1, is required endogenously by adult HSC to maintain self-renewal capacity and proper resilience of the hematopoietic system during regeneration.

PEDF Can Rescue the Inhibition of Injured Endothelial Cells on Hematopoietic Stem Cell Expansion Ex Vivo

Introduction: Endothelial cells (ECs) provide a fertile niche for hematopoietic stem cell (HSC) maintenance, differentiation, and migration.Several studies have indicated that bone marrow (BM) vascular niche was impaired after HSC transplantation and severely inhibited hematopoietic reconstruction. Pigment epithelium-derived factor (PEDF) is an important potential cytoprotection and therapeutic agent for injured cells. The direct role of the injured endothelial cells on hematopoietic stem cells and whether PEDF has protective effect in this system remain unknown. This study aims to observe the influence of enjured ECs on HSCs and to explore the role of PEDF in endothelial-HSC coculture system. Methods: Injury of Endothelial cells by two important preparative regimenconditioning radiation and Busulfan respectively was evaluated with CCK8 assay. The expression of endothelial tight junctions(TJs),adherent junctions related molecules and endothelial to Mesenchymal Transition molecules such as ZO-1, Occludin,VE-cadherin, ICAM, α-SMA, CD31 and VCAM were detected by RT-qPCR, flow cytometry, immunofluorescence and western blot. The effects of injured endothelial cells on HSC self-renewal, differentiation, cell cycle and apoptosis were evaluated by flow cytometry, photography, viable cell count and clone formation assay. Hematopoiesis regulation factors SCF, IL-6, TGF-β and TNF-α were detected by ELISA. The protective effect of PEDF was also explored. Results: Both radiation and Busulfan could decrease cell viability of endothelial cells. The expression level of ZO-1, Occludin, VE-cadherin, ICAM, CD31 and VCAM were decreased and α-SMA was increased when EC exposed to radiation or Busulfan suggesting endothelial activation, impaired EC permeability and endothelial to Mesenchymal Transition after EC injured. Compared with normal endothelial cells and hematopoietic stem cell co-culture group, the HSC% of injured endothelial cells and hematopoietic stem cells co-cultured group were significantly decreased, the cell colony formation ability was decreased, the proportion of mature cells increased, and the damage of endothelial cells could not maintain the characteristics of HSC, weakened the self-renewal and multidirectional differentiation potential of HSC and promoted the maturation of HSC. After the administration of PEDF, endothelial to Mesenchymal Transition of EC was suppressed and the EC permeability was improved. Most importantly, the proportion of HSC was significantly increased, and the proportion of mature cells decreased in the coculture system. Conclusion: Injured endothelial cells can inhibit proliferation of hematopoietic stem cells, self-renewal and promote HSC differentiation. PEDF could ameliorate endothelial injury and promote HSC expansion by suppressing endothelial-mesenchymal transition and protecting TJs and AJs. Disclosures No relevant conflicts of interest to declare.

Electron Transport Chain Complex II Sustains High Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells

The role of mitochondria in the fate determination of hematopoietic stem and progenitor cells (HSPCs) is not solely limited to the switch from glycolysis to oxidative phosphorylation, but also involves alterations in mitochondrial features and properties, including mitochondrial membrane potential (ΔΨmt). Several research groups have used mitochondrial dyes and have showed that long term multi-lineage reconstitution is enriched in low ΔΨmt fraction. However, hematopoietic stem cells (HSCs) exhibit higher pump activity than mature populations, and this causes the enhanced extrusion of mitochondrial dyes used for measuring ΔΨmt, such as tetramethylrhodamine methyl ester (TMRM), which in turn can lead to biased results (Bonora M. et al, 2018). In this study, while considering the activity of xenobiotic efflux pumps in HSCs, we have assessed the equilibrium between electron transport chain (ETC) complexes and ATP production in order to elucidate the mechanism that sustain mitochondrial membrane potential in HSPCs. We first used flow analysis of HSCs and other bone marrow populations, stained by TMRM in presence of Verapamil, an efflux pump inhibitor, to show a downward trend in ΔΨmt along with hematopoietic differentiation. To validate high ΔΨmt as a key feature of HSCs, we measured Ki67 positivity to assess whether ΔΨmt is associated with cell cycle quiescence in HSPCs. When Lin-Sca-1+c-Kit+ (LSK) cells were separated into two fractions, based on their TMRM intensity, we found that the percentage of Ki67+ cells in LSK-High was lower than the one in LSK-Low, and were comparable to CD150+CD48- HSC-enriched fraction. Consistently, phenotypic HSCs preferentially reside in the TMRM high population, with less Ki67 positivity. Since ΔΨmt levels in the cells is determined by the balance between proton pumping (by ETC) and proton flow (by ATP synthase/complex V), we next assessed ETC complexes. The expression of ATP5A, a key subunit of complex V, and of NDUFV1, a subunit of complex I, were particularly weak in HSPCs and drastically increased following differentiation process, while no differences were detected in complex II subunit SDHA expression between HSCs and mature populations. Likewise, the activity of complex I increased following differentiation process, while the activity of complex II remained stable among HSC, LSK, and Lin− fractions. Interestingly, when the respective ratios of complex I and II to complex V were calculated, compared to complex I, a significantly higher ratio of complex II: complex V was found in HSPCs. Collectively, these data support the hypothesis that HSPCs have low proton flow comparing mature populations, but similar proton pumping activity, especially due to complex II, which finally results in a higher ΔΨmt. In order to deeply investigate the contribution of complex I, II and III to sustain ΔΨmt, the reduction of TMRM intensity after the administration of low dosages of their specific inhibitors (Rotenone, TTFA and Antimycin A, respectively) was analyzed. The reduction of TMRM intensity by Rotenone was observed in the committed cells, and the addition of Antimycin A led to a drop in TMRM intensity in all hematopoietic lineages. Critically, complex II inhibition by TTFA caused a substantial decrease of ΔΨmt, particularly in HSPCs. Finally, we investigated the functional importance of each ETC complex in HSCs, founding that TTFA, but not Rotenone, caused a reduction in in vitro colony-replating capacity, and a similar effect was observed after administration of Antimycin A. Altogether this study highlights complex II as a key regulator of ΔΨmt in HSPCs and suggests the distinct roles of complex I and complex II in hematopoiesis. Further characterization of the precise mechanisms regulating mitochondrial controls in HSCs will contribute to a better understanding of an active role of mitochondria in HSC homeostasis. Disclosures No relevant conflicts of interest to declare.

"Hierarchy" and "Holacracy"; A Paradigm of the Hematopoietic System.

The mammalian hematopoietic system has long been viewed as a hierarchical paradigm in which a small number of hematopoietic stem cells (HSCs) are located at the apex. HSCs were traditionally thought to be homogeneous and quiescent in a homeostatic state. However, recent observations, through extramedullary hematopoiesis and clonal assays, have cast doubt on the validity of the conventional interpretation. A key issue is understanding the characteristics of HSCs from different viewpoints, including dynamic physics and social network theory. The aim of this literature review is to propose a new paradigm of our hematopoietic system, in which individual HSCs are actively involved.

Harnessing Hematopoietic Stem Cell Low Intracellular Calcium Improves Their Maintenance InVitro.

The specific cellular physiology of hematopoietic stem cells (HSCs) is underexplored, and their maintenance invitro remains challenging. We discovered that culture of HSCs in low calcium increased their maintenance as determined by phenotype, function, and single-cell expression signature. HSCs are endowed with low intracellular calcium conveyed byelevated activity of glycolysis-fueled plasma membrane calcium efflux pumps and a low-bone-marrow interstitial fluid calcium concentration. Low-calcium conditions inhibited calpain proteases, which target ten-eleven translocated (TET) enzymes, of which TET2 was required for the effect of low calcium conditions on HSC maintenance invitro. These observations reveal a physiological feature of HSCs that can be harnessed to improve their maintenance invitro.

PS1554 CLINICAL PROFILE AND OUTCOMES IN PATIENTS RECEIVING CULTURE POSITIVE HEMATOPOIETIC STEM CELL HARVEST: RETROSPECTIVE STUDY FROM AN ONCOLOGY UNIT IN A TERTIARY CARE CENTER IN INDIA

Background:Hematopoietic stem cells (HSC) may act as a source of infection for the recipient due to manipulation at multiple levels from collection to infusion. Due to the high risk of contamination cultures are usually taken during multiple steps. The clinical significance of microbial contamination of HSC on the post‐transplant course and the role of prophylactic antibiotics is relatively unknown.Aims:The main objective of our study is to investigate the prevalence of microbial contamination of hematopoietic stem cell at our center and to assess its impact on the post‐transplant course.Methods:Records of all patients admitted in the bone marrow transplantation unit of a tertiary care center in India between January 2014 and December 2018 were analyzed after taking approval from the Institute's ethical committee. Clinical profile of patients including diagnosis, the indications of the transplant, conditioning regimen, details of cryopreservation, details of HSC culture and sensitivity were recorded. Details of episodes of febrile neutropenia including the day of onset, duration, number of febrile episodes, the focus of infection, use of prophylactic antibiotics, antibiotics administered, neutrophil engraftment, platelets engraftment were recorded.Results:Out of the 1307 stem cell harvests from 503 patients sent for culture, 17 harvests (1.3%) were found to have a culture positive report. Baseline characteristics of HSC culture‐positive patient are shown in Table 1. Sixteen of the seventeen patients had undergone autologous transplant. Multiple myeloma was most common indication of HSC transplant followed by Non‐Hodgkin Lymphoma (NHL). HSC harvests were cryopreserved in eight patients and duration of cryopreservation ranged from 7 to 34 days. Prophylactic ciprofloxacin, trimethoprim‐sulfamethoxazole, itraconazole, and acyclovir were used in all patient. Twelve of seventeen HSC cultures were positive at the time of infusion and five were positive at the time of harvest. The five HSC that were culture positive at the time of harvest were culture negative at the time of infusion. Gram‐positive organisms were isolated in six cultures and gram‐negative in rest. All patients developed febrile neutropenia post‐transplantation between day 1 and day 7. The median time of onset of fever was day +5 (1–7), the median duration of fever was 4 days (2–7), the median duration of antibiotic use was 11 days (9–16). Empiric treatment for febrile neutropenia in 13 out of 17 patients also covered for the organism grown in the HSC culture. Eight of these patients responded appropriately to the initial treatment while nine patient required the addition of other antibiotics. Only one patient had blood culture positivity post‐transplant and culture yielded the same organism as HSC culture. Median day for neutrophil engraftment was 11 days (9–16), the median day for platelet engraftment was 14 days (8–25) and median day for the duration of hospitalization was 15 days (12–78). All patients were alive at day 100 of transplant. Two patient which had prolonged stay, one had urinary tract infection post neutrophil engraftment; the second patient was the case of B‐ALL had acute GVHD, CMV colitis and VOD.Summary/Conclusion:Our study shows that there is no effect of culture‐positive HSC on transplant‐related outcomes in terms of the fever onset, duration of fever, neutrophil and platelet engraftment, duration of hospitalization and day 100 mortality. There is a need for larger prospective studies to understand the clinical relevance of such contaminations and the need for antibiotic prophylaxis in such cases.image

Promoted Proliferation of Hematopoietic Stem Cells Enabled by a Hyaluronic Acid/Carbon Nanotubes Antioxidant Hydrogel

AbstractReactive oxygen species (ROS) level is closely associated with the physiological function of hematopoietic stem cells (HSCs) in ex vivo culture systems. Previously developed hydrogels in use of HSCs culture have generally failed in considering their antioxidant property. In the present work, antioxidant hydrogels are constructed to evaluate the biological effect of ROS on HSCs proliferation by suppressing oxidative stress damage. Functionalized carbon nanotubes (CNTs), which act as potential antioxidants, are physically encapsulated in biocompatible hyaluronic acid (HA) hydrogels to achieve long‐term antioxidant activity. Consequently, these hybrid hydrogels exhibit enhanced physical properties and superior scavenging capability on oxides and peroxides compared to the pure HA hydrogel. The results indicate that ROS significantly inhibit the biological characteristics of HSCs. Nevertheless, the proliferation ability and pluripotency are dramatically improved when the culture system is supplied with the antioxidant hydrogel, revealing that the CNT‐incorporated hydrogel can effectively relieve oxidative stress response in HSCs and reduce apoptosis from ROS. Therefore, the HA/CNT hydrogel can provide a novel strategy to establish an artificial microenvironment with a low ROS level for HSCs proliferation.

YY1 Controls Hematopoietic Stem Cell Quiescence By Repressing Cohesin Expression

Hematopoietic stem cells (HSCs) are undifferentiated, self-renewing, pluripotent cells that have the capacity to differentiate into all mature lineage-specific cells in adult blood. Adult HSCs can remain in a quiescent state for a prolonged time, and quiescence is a fundamental characteristic of HSCs in adult bone marrow. Thus, the cell cycle must be precisely regulated. Yin Yang 1 (YY1) is a multifunctional transcription factor and Polycomb Group Protein (PcG) that is important for embryonic development, adult hematopoiesis, cell proliferation and maintaining higher-order chromosomal structure. We have generated YY1 conditional knockout mice (Yy1f/f Mx1-Cre) and showed that Yy1 deficient HSCs fail to self-renewal and had disrupted HSCs quiescence. Stem cell factor (SCF)/c-Kit signaling, a critical regulatory pathway in HSC development, is significantly downregulated in Yy1-/- HSCs. Interestingly, YY1 regulation of HSCs self-renewal and quiescence is independent on its PcG domain/function. Instead, YY1 occupied at Smc3 promoter area and repressed Smc3 expression. In Yy1-/-HSCs, Smc3 expression was upregulated. SMC3 is a core component of cohesin protein complex and plays critical roles in HSC self-renewal, myeloid differentiation and leukemogenesis. To further dissect the underlying mechanisms by which YY1 regulates SMC3 expression in HSCs, we have generated conditional knockout mice with YY1 homozygous deletion and SMC3 heterozygous deletion (Yy1f/f Smc3f/+Mx1-Cre). In Yy1-/- Smc3+/- bone marrow cells, SMC3 expression was normalized to the wild-type level. In adult bone marrow cells, YY1 physically interacted with cohesin complex proteins through its zinc finger domain. By analyzing the YY1, SMC1A and SMC3 ChIP-Seq database, our study showed that YY1 and cohesin co-occupied at promoter areas of genes that are critical for cell metabolism. Evidence from previous study showed that impaired metabolism, including increased reactive oxygen species (ROS) and decreased mitochondrial function, can cause defect in stem cell self-renewal and quiescence. In Yy1-/- Smc3+/-HSCs, cell quiescence was restored although HSC self-renewal was still impaired, indicates that YY1 and SMC3 may control HSC cell quiescence via regulating genes critical for cell metabolism. Our study identified YY1 as the first transcription factor that regulates expression of cohesin complex component SMC3. We are currently further dissecting underlying mechanisms and functional significances of metabolic pathways regulated by YY1-SMC3 axis in HSCs. DisclosuresLevine:Imago: Equity Ownership; Isoplexis: Equity Ownership; Janssen: Consultancy, Honoraria; Gilead: Honoraria; Epizyme: Patents & Royalties; Loxo: Consultancy, Equity Ownership; C4 Therapeutics: Equity Ownership; Roche: Consultancy, Research Funding; Prelude: Research Funding; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Novartis: Consultancy.

Low Intracellular Calcium is an Essential Feature of Hematopoietic Stem Cells

The specific cellular physiology of hematopoietic stem cells (HSCs) is underexplored and their maintenance in vitro remains challenging. We discovered that culture of HSC in low calcium increased their maintenance as determined by phenotype, function and single cell expression signature. HSCs are endowed with low intracellular calcium conveyed by elevated activity of glycolysis-fueled plasma membrane calcium efflux pumps and a low bone marrow interstitial fluid calcium concentration. HSC maintenance in low calcium conditions was mediated by inhibition of calpain proteases. Consistent with the fact that TET enzymes are targets of calpains, low calcium increased TET protein expression and 5-hydroxymethyl cytosine deposition and maintained TET2-asssociated single cell gene signature. Deletion of the TET homologue required for normal HSC function and identity, TET2, rendered HSC impervious to the effect low calcium conditions. These observations reveal a physiological feature of HSCs than can be harnessed to improve their maintenance in vitro.

Differences in Cell Cycle Status Underlie Transcriptional Heterogeneity in the HSC Compartment

Hematopoietic stem cells (HSCs) are considered a heterogeneous cell population. To further resolve the HSC compartment, we characterized a retinoic acid (RA) reporter mouse line. Sub-fractionation of the HSC compartment in RA-CFP reporter mice demonstrated that RA-CFP-dim HSCs were largely non-proliferative and displayed superior engraftmentpotential in comparison with RA-CFP-bright HSCs.Gene expression analysis demonstrated higher expression of RA-target genes in RA-CFP-dim HSCs, in contrast to the RA-CFP reporter expression, but both RA-CFP-dim and RA-CFP-bright HSCs responded efficiently to RA invitro. Single-cell RNA sequencing (RNA-seq) of >1,200 HSCs showed that differences in cell cycle activity constituted the main driver of transcriptional heterogeneity in HSCs. Moreover, further analysis of the single-cell RNA-seq data revealed that stochastic low-level expression of distinct lineage-affiliated transcriptional programs is a common feature of HSCs. Collectively, this work demonstrates the utility of the RA-CFP reporter line as a tool for the isolation of superior HSCs.