Divergent Levels of CD112 and INKA1 Define a Subset of Human Hematopoietic Stem Cells That Resists Regenerative Stress to Preserve Stemness

The first insight into the complexity of post-transplant in vivo dynamics of hematopoietic stem cells (HSC) in humans was only recently showcased by examining longitudinal clonal contributions in gene therapy patients. Initial blood reconstitution was achieved by short-term (ST-)HSC, but in the longer-term hematopoiesis originated from long-term (LT-)HSC that only became recruited after a latency phase of 1-2 years (Scala et al 2018). Thus, deciphering the mechanisms governing how LT-HSC might resist transplant mediated activation and/or respond to the varying hematopoietic demands that occur during homeostasis would be an important goal for improving HSC cell therapies. We previously linked INKA1(=C3orf54/FAM212A) mediated PAK4 inhibition and reduced H4K16 acetylation (H4K16ac) to quiescence in human leukemia stem cells (Kaufmann, Blood 2019). Here, we interrogate the role of this signaling axis in normal human hematopoiesis. Immunostaining revealed distinct subsets defined by the dichotomy of INKA1 and H4K16ac within cord blood ST- and LT-HSC, mechanistically supported by BioID, chromatin fiber analysis and PLA data showing INKA1 interacting with the H4K16 deacetylase, SIRT1. Among quiescent LT-HSC, we found that the INKA1high fraction (~10 % of LT-HSC) had the lowest CDK6 levels and represented LT-HSC in an alternative state of quiescence. We then used protein interaction (BioID) data to show that a shared interactor of INKA1 and PAK4, CD112 could be used to sort for the subset of CD112low LT-HSC that was in this alternatively quiescent LT-HSC state (H4K16aclow, CDK6low, CellROXlow). Compared to primary cells, culture attenuates the strict dichotomy of INKA1 and H4K16ac but only in cells in which colocalization of PAK4 with both occurred, suggesting PAK4 interferes with SIRT1-INKA1 interaction thereby permitting H4K16 acetylation. In vitro time course analysis showed that H4K16ac and PAK4 levels preceded the upregulation of the G0-exit marker CDK6 implicating a role in cell cycle priming. Pseudo-time scRNAseq analysis for INKA1high versus PAK4high, CDK6high and CD112high expression in mobilized peripheral blood (modeling in vivo HSC activation) showed enrichment of early or late diffusion indicative of quiescent versus primed cell status, respectively. Strikingly, in xenograft assays CD112low LT-HSC exhibited delayed engraftment kinetics with higher secondary repopulation capacity than faster repopulating CD112high LT-HSC reflecting the subset of LT-HSC that resist early activation. Similarly, INKA1-OE or PAK4 knock-down in vivo resulted in an early restraint in engraftment levels (@4 w), whereas 20 w engraftment reached control levels. When measured in secondary transplant assays the HSC frequency was 4 to 8-fold higher in the groups that showed this early restraint. Thus, resisting early activation (latency) preserves the regenerative potential of such HSC. In vivo 5-Fluorouracil treatment at week 4 accelerated latency exit and further increased HSC frequency of INKA1-OE cells while abolishing serial transplantation potential of controls. Hence, the induction of proliferative stress via creating acute hematopoietic demand is able to lift the INKA1-OE imposed restraint resulting in increased hematopoietic output while also promoting HSC self-renewal. Collectively, our data decipher the molecular intricacies underlying HSC heterogeneity and self-renewal regulation and point to latency as an orchestrated physiological response that integrates quiescence control with HSC fate choices to preserve a stem cell reservoir. Disclosures Takayanagi: Kirin Holdings Company, Ltd: Current Employment. Dick:Bristol-Myers Squibb/Celgene: Research Funding.

Download Full-text

Cripto Selectively Expands a Distinct Population of Hematopoietic Stem Cells Expressing the Cell Surface Receptor GRP78 and Strongly Induces An Immature Phenotype In Vivo After Ex Vivo Culture

Blood ◽

10.1182/blood.v116.21.405.405 ◽

2010 ◽

Vol 116 (21) ◽

pp. 405-405

Author(s):

Kenichi Miharada ◽

Göran Karlsson ◽

Jonas Larsson ◽

Emma Larsson ◽

Kavitha Siva ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Distinct Population ◽

Self Renewal ◽

Hematopoietic Stem ◽

Total Bone Marrow

Abstract Abstract 405 Cripto is a member of the EGF-CFC soluble protein family and has been identified as an important factor for the proliferation/self-renewal of ES and several types of tumor cells. The role for Cripto in the regulation of hematopoietic cells has been unknown. Here we show that Cripto is a potential new candidate factor to increase self-renewal and expand hematopoietic stem cells (HSCs) in vitro. The expression level of Cripto was analyzed by qRT-PCR in several purified murine hematopoietic cell populations. The findings demonstrated that purified CD34-KSL cells, known as highly concentrated HSC population, had higher expression levels than other hematopoietic progenitor populations including CD34+KSL cells. We asked how Cripto regulates HSCs by using recombinant mouse Cripto (rmCripto) for in vitro and in vivo experiments. First we tested the effects of rmCripto on purified hematopoietic stem cells (CD34-LSK) in vitro. After two weeks culture in serum free media supplemented with 100ng/ml of SCF, TPO and 500ng/ml of rmCripto, 30 of CD34-KSL cells formed over 1,300 of colonies, including over 60 of GEMM colonies, while control cultures without rmCripto generated few colonies and no GEMM colonies (p<0.001). Next, 20 of CD34-KSL cells were cultured with or without rmCripto for 2 weeks and transplanted to lethally irradiated mice in a competitive setting. Cripto treated donor cells showed a low level of reconstitution (4–12%) in the peripheral blood, while cells cultured without rmCripto failed to reconstitute. To define the target population and the mechanism of Cripto action, we analyzed two cell surface proteins, GRP78 and Glypican-1, as potential receptor candidates for Cripto regulation of HSC. Surprisingly, CD34-KSL cells were divided into two distinct populations where HSC expressing GRP78 exhibited robust expansion of CFU-GEMM progenitor mediated by rmCripto in CFU-assay whereas GRP78- HSC did not respond (1/3 of CD34-KSL cells were GRP78+). Furthermore, a neutralization antibody for GRP78 completely inhibited the effect of Cripto in both CFU-assay and transplantation assay. In contrast, all lineage negative cells were Glypican-1 positive. These results suggest that GRP78 must be the functional receptor for Cripto on HSC. We therefore sorted these two GRP78+CD34-KSL (GRP78+HSC) and GRP78-CD34-KSL (GRP78-HSC) populations and transplanted to lethally irradiated mice using freshly isolated cells and cells cultured with or without rmCripto for 2 weeks. Interestingly, fresh GRP78-HSCs showed higher reconstitution than GRP78+HSCs (58–82% and 8–40%, p=0.0038) and the reconstitution level in peripheral blood increased rapidly. In contrast, GRP78+HSC reconstituted the peripheral blood slowly, still at a lower level than GRP78-HSC 4 months after transplantation. However, rmCripto selectively expanded (or maintained) GRP78+HSCs but not GRP78-HSCs after culture and generated a similar level of reconstitution as freshly transplanted cells (12–35%). Finally, bone marrow cells of engrafted recipient mice were analyzed at 5 months after transplantation. Surprisingly, GRP78+HSC cultured with rmCripto showed higher reconstitution of the CD34-KSL population in the recipients' bone marrow (45–54%, p=0.0026), while the reconstitution in peripheral blood and in total bone marrow was almost the same. Additionally, most reconstituted CD34-KSL population was GRP78+. Interestingly freshly transplanted sorted GRP78+HSC and GRP78-HSC can produce the GRP78− and GRP78+ populations in the bone marrow and the ratio of GRP78+/− cells that were regenerated have the same proportion as the original donor mice. Compared to cultured cells, the level of reconstitution (peripheral blood, total bone marrow, HSC) in the recipient mice was almost similar. These results indicate that the GRP78 expression on HSC is reversible, but it seems to be “fixed” into an immature stage and differentiate with lower efficiency toward mature cells after long/strong exposure to Cripto signaling. Based on these findings, we propose that Cripto is a novel factor that maintains HSC in an immature state and may be a potent candidate for expansion of a distinct population of GRP78 expressing HSC. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Polycomb Group Gene rae28 Is Required for Sustaining Activity of Hematopoietic Stem Cells

Journal of Experimental Medicine ◽

10.1084/jem.20011911 ◽

2002 ◽

Vol 195 (6) ◽

pp. 759-770 ◽

Cited By ~ 125

Author(s):

Hideaki Ohta ◽

Akihisa Sawada ◽

Ji Yoo Kim ◽

Sadao Tokimasa ◽

Seiji Nishiguchi ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Polycomb Group ◽

Self Renewal ◽

Hematopoietic Stem ◽

Polycomb Group Genes ◽

Hematopoietic Activity

The rae28 gene (rae28), also designated as mph1, is a mammalian ortholog of the Drosophila polyhomeotic gene, a member of Polycomb group genes (PcG). rae28 constitutes PcG complex 1 for maintaining transcriptional states which have been once initiated, presumably through modulation of the chromatin structure. Hematopoietic activity was impaired in the fetal liver of rae28-deficient animals (rae28−/−), as demonstrated by progressive reduction of hematopoietic progenitors of multilineages and poor expansion of colony forming units in spleen (CFU-S12) during embryonic development. An in vitro long-term culture-initiating cell assay suggested a reduction in hematopoietic stem cells (HSCs), which was confirmed in vivo by reconstitution experiments in lethally irradiated congenic recipient mice. The competitive repopulating units (CRUs) reflect HSCs supporting multilineage blood-cell production. CRUs were generated, whereas the number of CRUs was reduced by a factor of 20 in the rae28−/− fetal liver. We also performed serial transplantation experiments to semiquantitatively measure self-renewal activity of CRUs in vivo. Self-renewal activity of CRUs was 15-fold decreased in rae28−/−. Thus the compromised HSCs were presumed to reduce hematopoietic activity in the rae28−/− fetal liver. This is the first report to suggest that rae28 has a crucial role in sustaining the activity of HSCs to maintain hematopoiesis.

Download Full-text

Stromal Cell Regulation of Murine Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v116.21.1566.1566 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1566-1566

Author(s):

Stefan Wohrer ◽

Keegan Rowe ◽

Heidi Mader ◽

Claudia Benz ◽

Michael R Copley ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Stromal Cell ◽

Cultured Cells ◽

Conflicts Of Interest ◽

Self Renewal ◽

Hematopoietic Stem ◽

Daughter Cells

Abstract Abstract 1566 Recent advances in purifying murine hematopoietic stem cells (HSCs) to near homogeneity (>20%) have made it possible to analyze their in vivo clonal growth, self-renewal and differentiation properties over prolonged periods and the effects of various manipulations on these key functional parameters. However, conditions that allow genetically unaltered HSCs to maintain their original functional properties over equivalent periods of prolonged proliferation in vitro have not yet been identified. Since initial studies showed that the UG26 stromal cell could support murine HSC maintenance for limited periods, we first asked whether the addition of cytokines that also maintain HSCs for short periods might synergize with UG26 cells to enable HSC expansion to occur. Limiting dilution transplants that used a 6-month read-out of reconstituted blood elements (>1%) showed that the addition of 100 ng/ml Steel Factor (SF) and 20 ng/ml IL-11 to cultures containing UG26 cells and single purified (50%) HSCs (EPCR+CD150+CD48-, ESLAM cells) consistently stimulated a 3–5 fold HSC expansion after 7 days (3 expts). Furthermore, the effect of the UG26 cells could be replaced by UG26 conditioned medium (CM) and, in the presence of the CM+SF/IL-11 cocktail, the HSCs showed sustained longterm in vivo lympho-myeloid reconstituting activity in both primary and secondary recipients. Under these conditions, every ESLAM cell isolated proliferated several times within 7 days, but individual analysis of paired daughter cells showed that most first divisions (13/42) were, nevertheless, asymmetrical in terms of the numbers and types of different lineages produced by each of the 2 daughter cells for at least 4 months, although occasional evidence of symmetry was obtained (2/42 divisions). Interestingly, these first divisions showed a biphasic curve with 75% of the cells dividing before and 25% after 48 hours - the late dividers being more highly enriched for HSCs (95% vs 20%). We next asked whether TGF-β might be an important factor in UG26 CM, since UG26 cells exert a strong cell cycle inhibitory effect, and produce abundant TGF-beta. Accordingly, we next analyzed the effect of adding a neutralizing anti-TGF-β antibody or replacing the CM with TGF-β in the same type of single HSC cultures by tracking the survival and division kinetics of the cells as well as measuring the repopulating activity of their in vitro progeny present after 7 days. Strikingly, the addition of anti-TGF-β to the CM+SF/IL-11 supplemented HSC cultures eliminated the late wave of first cell divisions and caused an accompanying loss of myeloid reconstituting ability in recipients transplanted with the cultured cells. Conversely, replacement of the CM with TGF-β restored a biphasic division kinetics curve to cultures supplemented with SF/IL-11 but no CM. However, this did not protect against the early 50% loss of cells by apoptosis. These findings provide evidence of a new role of TGF-β in preserving the integrity of HSC functionality in vitro, but suggest a requirement for other types of factors released by certain stromal cells to achieve sustained symmetrical HSC self-renewal in vitro. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Interferon-Gamma Impairs the Self-Renewal of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.2351.2351 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2351-2351

Author(s):

Alexander M. de Bruin ◽

Berend Hooibrink ◽

Martijn A. Nolte

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Interferon Gamma ◽

Chimeric Mice ◽

Self Renewal ◽

Hematopoietic Stem ◽

The Impact

Abstract Abstract 2351 Regulation of hematopoiesis during stress situations, such as bacterial or viral infections, is crucial for the maintenance of sufficient numbers of cells in the blood. It has become clear that activated immune cells provide such feedback signals to the bone marrow. An important mediator in this respect is the pro-inflammatory cytokine Interferon-gamma (IFNγ), which is produced in the bone marrow by activated T cells during the course of an infection. As such, we have previously shown that T cell-derived IFNγ can directly influence the output of myeloid and erythroid cells. To address whether IFNγ can also influence the function of hematopoietic stem cells (HSCs), we cultured highly purified HSCs from murine bone marrow with or without IFNγ and found that IFNγ strongly reduced the absolute number of HSCs in these cultures, both phenotypically and functionally. We confirmed that the functional impact of IFNγ was due to a direct effect on HSCs and not mediated by more differentiated progenitors. In addition, IFNγ does not directly influence the quiescent state of purified HSC, nor their cell cycle entry. By labeling HSCs with CFSE, we found that IFNγ reduces HSC expansion in vitro by decreasing their proliferative capacity, but not their ability to differentiate. To investigate the impact of IFNγ on HSCs in vivo, we infected WT and IFNγ−/− mice with lymphocytic choriomeningitis virus (LCMV) and found that IFNγ severely impaired HSC recovery upon infection. Finally, to exclude indirect effects of IFNγ on other cell types we generated chimeric mice with bone marrow from both WT and IFNγR−/− mice. Infection of these mixed-chimeric mice with LCMV resulted in decreased recovery of WT HSCs, but not of IFNγR−/− HSCs in the same mouse, which formally demonstrates that IFNγ directly impairs the proliferation of HSCs in vivo. Based on these experiments we conclude that IFNγ reduces HSC self renewal both in vitro and in vivo. Importantly, we thereby challenge the current concept in literature that IFNγ would induce the proliferation of HSCs (Baldridge et al, Nature 2010). Our findings thus provide challenging new insight regarding the impact of immune activation on hematopoiesis and will contribute significantly to the scientific discussion concerning this process. Moreover, our data also provide an explanation for the occurrence of anemia and bone marrow failure in several human diseases in which IFNγ is chronically produced. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Steel factor responsiveness regulates the high self-renewal phenotype of fetal hematopoietic stem cells

Blood ◽

10.1182/blood-2006-08-037770 ◽

2007 ◽

Vol 109 (11) ◽

pp. 5043-5048 ◽

Cited By ~ 66

Author(s):

Michelle B. Bowie ◽

David G. Kent ◽

Michael R. Copley ◽

Connie J. Eaves

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Wild Type ◽

Developmentally Regulated ◽

Self Renewal ◽

Hematopoietic Stem ◽

Steel Factor ◽

Reduced Rate

Abstract Fetal hematopoietic stem cells (HSCs) regenerate daughter HSCs in irradiated recipients more rapidly than do adult HSCs. However, both types of HSCs divide in vitro with the same cell-cycle transit times, suggesting different intrinsically determined self-renewal activities. To investigate the mechanism(s) underlying these differences, we compared fetal and adult HSC responses to Steel factor (SF) stimulation in vitro and in vivo. These experiments were undertaken with both wild-type cells and W41/W41 cells, which have a functionally deficient c-kit kinase. In vitro, fetal HSC self-renewal divisions, like those of adult HSCs, were found to be strongly dependent on c-kit activation, but the fetal HSCs responded to much lower SF concentrations in spite of indistinguishable levels of c-kit expression. Fetal W41/W41 HSCs also mimicked adult wild-type HSCs in showing the same reduced rate of amplification in irradiated adult hosts (relative to fetal wild-type HSCs). Assessment of various proliferation and signaling gene transcripts in fetal and adult HSCs self-renewing in vitro revealed a singular difference in Ink4c expression. We conclude that the ability of fetal HSCs to execute symmetric self-renewal divisions more efficiently than adult HSCs in vivo may be dependent on specific developmentally regulated signals that act downstream of the c-kit kinase.

Download Full-text

Hematopoietic stem cells acquire survival advantage by loss of RUNX1 methylation identified in familial leukemia

Blood ◽

10.1182/blood.2019004292 ◽

2020 ◽

Vol 136 (17) ◽

pp. 1919-1932

Author(s):

Takayoshi Matsumura ◽

Ayako Nakamura-Ishizu ◽

Siva Sai Naga Anurag Muddineni ◽

Darren Qiancheng Tan ◽

Chelsia Qiuxia Wang ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Posttranslational Modifications ◽

Dominant Negative ◽

Survival Advantage ◽

Human Leukemia ◽

Hematopoietic Stem ◽

Genetic Ablation

Abstract RUNX1 is among the most frequently mutated genes in human leukemia, and the loss or dominant-negative suppression of RUNX1 function is found in myelodysplastic syndrome and acute myeloid leukemia (AML). How posttranslational modifications (PTMs) of RUNX1 affect its in vivo function, however, and whether PTM dysregulation of RUNX1 can cause leukemia are largely unknown. We performed targeted deep sequencing on a family with 3 occurrences of AML and identified a novel RUNX1 mutation, R237K. The mutated R237 residue is a methylation site by protein arginine methyltransferase 1, and loss of methylation reportedly impairs the transcriptional activity of RUNX1 in vitro. To explore the biologic significance of RUNX1 methylation in vivo, we used RUNX1 R233K/R237K double-mutant mice, in which 2 arginine-to-lysine mutations precluded RUNX1 methylation. Genetic ablation of RUNX1 methylation led to loss of quiescence and expansion of hematopoietic stem cells (HSCs), and it changed the genomic and epigenomic signatures of phenotypic HSCs to a poised progenitor state. Furthermore, loss of RUNX1 R233/R237 methylation suppressed endoplasmic reticulum stress–induced unfolded protein response genes, including Atf4, Ddit3, and Gadd34; the radiation-induced p53 downstream genes Bbc3, Pmaip1, and Cdkn1a; and subsequent apoptosis in HSCs. Mechanistically, activating transcription factor 4 was identified as a direct transcriptional target of RUNX1. Collectively, defects in RUNX1 methylation in HSCs confer resistance to apoptosis and survival advantage under stress conditions, a hallmark of a preleukemic clone that may predispose affected individuals to leukemia. Our study will lead to a better understanding of how dysregulation of PTMs can contribute to leukemogenesis.

Download Full-text

Murine Hematopoietic Stem Cells Require Akt1 and Akt2 for Long-Term Self-Renewal

Blood ◽

10.1182/blood.v112.11.502.502 ◽

2008 ◽

Vol 112 (11) ◽

pp. 502-502

Author(s):

Marisa M. Juntilla ◽

Vineet Patil ◽

Rohan Joshi ◽

Gary A. Koretzky

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Akt Signaling ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Murine hematopoietic stem cells (HSCs) rely on components of the Akt signaling pathway, such as FOXO family members and PTEN, for efficient self-renewal and continued survival. However, it is unknown whether Akt is also required for murine HSC function. We hypothesized that Akt would be required for HSC self-renewal, and that the absence of Akt would lead to hematopoietic failure resulting in developmental defects in multiple lineages. To address the effect of Akt loss in HSCs we used competitive and noncompetitive murine fetal liver-bone marrow chimeras. In short-term assays, Akt1−/−Akt2−/− fetal liver cells reconstituted the LSK compartment of an irradiated host as well or better than wildtype cells, although failed to generate wildtype levels of more differentiated cells in multiple lineages. When placed in a competitive environment, Akt1−/−Akt2−/− HSCs were outcompeted by wildtype HSCs in serial bone marrow transplant assays, indicating a requirement for Akt1 and Akt2 in the maintainance of long-term hematopoietic stem cells. Akt1−/−Akt2−/− LSKs tend to remain in the G0 phase of the cell cycle compared to wildtype LSKs, suggesting the failure in serial transplant assays may be due to increased quiesence in the absence of Akt1 and Akt2. Additionally, the intracellular content of reactive oxygen species (ROS) in HSCs is dependent on Akt signaling because Akt1−/−Akt2−/− HSCs have decreased ROS levels. Furthermore, pharmacologic augmentation of ROS in the absence of Akt1 and Akt2 results in an exit from quiescence and rescue of differentiation both in vivo and in vitro. Together, these data implicate Akt1 and Akt2 as critical regulators of long-term HSC function and suggest that defective ROS homeostasis may contribute to failed hematopoiesis.

Download Full-text

Prolonged self-renewal activity unmasks telomerase control of telomere homeostasis and function of mouse hematopoietic stem cells

Blood ◽

10.1182/blood-2010-11-319632 ◽

2011 ◽

Vol 118 (7) ◽

pp. 1766-1773 ◽

Cited By ~ 11

Author(s):

Sanja Sekulovic ◽

Vala Gylfadottir ◽

Irma Vulto ◽

Maura Gasparetto ◽

Yasmine Even ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Self Renewal ◽

Telomere Shortening ◽

Hematopoietic Stem ◽

Telomere Homeostasis ◽

And Function ◽

The Impact

Abstract Strategies for expanding hematopoietic stem cells (HSCs) could have significant utility for transplantation-based therapies. However, deleterious consequences of such manipulations remain unknown. Here we examined the impact of HSC self-renewal divisions in vitro and in vivo on their subsequent regenerative and continuing ability to sustain blood cell production in the absence of telomerase. HSC expansion in vitro was obtained using a NUP98-HOXA10hd transduction strategy and, in vivo, using a serial transplant protocol. We observed ∼ 10kb telomere loss in leukocytes produced in secondary mice transplanted with HSCs regenerated in primary recipients of NUP98-HOXA10hd-transduced and in vitro-expanded Tert−/− HSCs 6 months before. The second generation leukocytes also showed elevated expression of γH2AX (relative to control) indicative of greater accumulating DNA damage. In contrast, significant telomere shortening was not detected in leukocytes produced from freshly isolated, serially transplanted wild-type (WT) or Tert−/− HSCs, suggesting that HSC replication posttransplant is not limited by telomere shortening in the mouse. These findings document a role of telomerase in telomere homeostasis, and in preserving HSC functional integrity on prolonged self-renewal stimulation.

Download Full-text

Hematopoietic Stem Cells Fail to Regenerate In Vivo Following Inflammatory Stress

Blood ◽

10.1182/blood.v128.22.1472.1472 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1472-1472

Author(s):

Ruzhica Bogeska ◽

Paul Kaschutnig ◽

Stella Paffenholz ◽

Julia Maassen ◽

Jan-Philipp Mallm ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Research Funding ◽

Post Transplantation ◽

Self Renewal ◽

Hematopoietic Stem ◽

Fold Reduction

Abstract An often-cited defining property of hematopoietic stem cells (HSCs) is their extensive or unlimited in vivo self-renewal capacity. We have recently described a novel mouse disease model forFanconi anemia, in which serial challenge with pro-inflammatory agonists that mimic infection, such aspolyinosinic:polycytidylic acid (pI:C), results in HSC attrition followed by a highly penetrant severe aplastic anemia, closely recapitulating the disease in patients (Walter et al., 2015, Nature). In order to explore the broader implications of these findings in the context of HSC self-renewal, we conducted apI:Cdose escalation regimen using standard C57BL6 mice. A single injection withpI:Cprovoked transient peripheral blood (PB)cytopenias, with the recovery of mature blood cell numbers correlating with HSCs being forced into active cell cycle. Injection with 1-3 rounds ofpI:C(1-3 x 8 injections) led to no discernable sustained impact on blood production as, at 5 weeks post-treatment, PB frequencies were in the normal range, as were the absolute numbers of HSCs and all progenitor compartments in the bone marrow (BM), as determined by flowcytometry. However, in vitro analysis of the proliferation and differentiation potential of 411 individual sorted long-term (LT)-HSCs 5 weeks after 3 rounds of pI:C challenge, revealed a decrease in the frequency of LT-HSCs able to generate progeny in vitro (1.6-fold reduction, p<0.05), and a 2-fold reduction in the total number of progeny produced per HSC, which was even more pronounced inmultilineage potential clones (2.6-fold decrease, p<0.0001) compared touni- or bi-lineage clones. In line with this data, competitive repopulation assays demonstrated a progressive depletion of functional HSC numbers with increasing rounds ofpI:C treatment, with a 1.8, 3.4 and 15.3-fold decrease in donorchimerism across all lineages at 6 months post-transplantation (p<0.01) following 1, 2 or 3 rounds ofpI:C treatment, respectively. Notably, robust engraftment (up to 65% donorchimerism, 6 months post-transplantation, p<0.01) was achieved when mice exposed to 3 rounds ofpI:C treatment were used as a recipient for non-treated BM cells in the absence of any irradiation conditioning, while engraftment was always <1% when non-treated controls were used as recipients. This excludes the possibility that the observed progressive depletion of functional HSCs was the result of artifacts associated with a compromised niche or the non-physiologic stress imposed on donor cells during transplantation. In order to test the kinetics of HSC recovery following HSC challenge, BM was harvested from mice at either 5, 10 or 20 weeks after treatment with 3 rounds of pI:C, and both competitive and limiting dilution transplantation assays (Table 1) were used to quantify HSC frequencies. Surprisingly, both assays demonstrated that HSCs failed to regenerate at all following pI:Cchallenge, directly contradicting the canonical view that HSCs possess extensive self-renewal capacity in vivo. The physiologic relevance of this observation was illustrated when we measured the hematologic parameters of aged mice that had been exposed to chronicpI:C treatment in early to mid-life. Although these mice had normal PB counts at 4 weeks post-treatment, at 2 years of age, peripheral bloodcytopenias and bone marrow aplasia became evident (Table 2), recapitulating clinically relevant features of non-malignant aged human hematopoiesis that are never seen in standard laboratory mice. Together, these data suggest that functional HSCs can be progressively and irreversibly depleted in response to environmental agonists, such as infection and inflammation, which force HSCs to reconstitute mature blood cells consumed by such stimuli. This model has clear implications relating to the role of adult stem cells in tissue maintenance and regeneration during ageing, and how stress agonists that are absent in most laboratory animal models, but would be ubiquitous in the wild, are likely key mediators of age-associated disease pathologies. Disclosures Frenette: PHD Biosciences: Research Funding; Pfizer: Consultancy; GSK: Research Funding.

Download Full-text

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15666200225091339 ◽

2020 ◽

Vol 15 ◽

Author(s):

Fatima Aerts-Kaya

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Ex Vivo ◽

Stress Conditions ◽

Stress Factors ◽

Hematopoietic Stem ◽

Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

Download Full-text