Long-term Bone Marrow Reconstitution Research Articles

Characterization of Hematopoietic Stem Cell Diversity Using Spatiotemporal Quantitative Phase Imaging

Cells undergo continual transformations, never retaining their original state. Hematopoietic stem cells (HSCs), at the apex of the hematopoietic hierarchy, are not exempt from this dynamic process. In recent years, the advent of single-cell analysis techniques such as scRNAseq has progressively unveiled the heterogeneity within cell populations previously presumed to be homogeneous. However, these techniques are limited to evaluating the cellular state at a single temporal point based on snapshot analysis, thus failing to account for temporal changes and dynamic behavior of cells. Therefore, the true diversity inherent to the ever-changing HSCs remains uncertain. In this study, we comprehensively elucidate HSC diversity, including temporal changes, by integrating our recently established ex vivo HSC expansion techniques (Wilkinson et al., Nature, Becker et al., Cell Stem Cell), single-cell quantitative phase imaging (QPI), and post-transplantation dynamics analysis. For live imaging, HSCs were harvested from hepatic leukemia factor ( Hlf)-tdTomato reporter mice, which specifically marks long-term HSCs. Index sorted single Hlf-tdTomato + HSC were expanded with a single-HSC culture system and monitored by QPI fluorescence time-lapse imaging. Surprisingly, even though the cells were derived from the same HSC fraction, significant variations in mitotic rate, motility, morphological changes, and Hlf expression levels were observed at the single-cell level. Stemness did not decrease stepwise over time, but always exhibited continuous changes. Thus, analysis including the temporal dimension illuminated the infinitely branched nature of HSC diversity. Interestingly, Hlf expression level correlated with the dynamic behavior of HSCs in the expansion culture system. Hlf-high HSCs exhibited a slow division rate, small, rounded morphology and low motility, while Hlf-low HSCs actively divided and differentiated, displayed larger morphological size with low sphericity, and exhibited high motility. Furthermore, spatiotemporal dynamics analysis of Hlf-high/low HSCs after transplantation, employing the highly sensitive in vivo imaging system Akaluc bioluminescence imaging (AkaBLI), revealed that Hlf-high HSCs initiate systemic hematopoiesis slowly, while Hlf-low HSCs expanded quickly, characterized by short-term explosive hematopoiesis in the spleen. As expected, only Hlf-high HSCs had long-term bone marrow reconstitution ability. Thus, based on the expression level of Hlf, it is possible to predict the dynamic behavior of the cells in the expansion medium and in the post-transplant body. Next, we investigated whether Hlf expression level could be predicted based on the kinetic features of cells during HSC ex vivo expansion. Interestingly, multidimensional analysis based on kinetic features extracted from time-lapse imaging showed multiple clusters, each linked to Hlf expression levels. In human HSCs, the dynamic diversity of HSCs was also observed in our recently established ex vivo expansion system (Sakurai et al., Nature). Notably, the kinetic features of human HSCs differed from those of mouse HSCs. Transplantation of human HSCs exhibiting specific morphological characteristics showed long-term reconstitution ability. Thus, classification of HSCs based on kinetic features facilitated enrichment of functional HSCs. This study demonstrates the importance of characterizing cells, including the temporal dimension, and illustrates the existence of infinite dynamic individuality in mouse and human HSCs.

Single-cell lineage tracing unveils a role for TCF15 in haematopoiesis.

SummaryBone marrow transplantation therapy relies on the life-long regenerative capacity of haematopoietic stem cells (HSCs)1,2. HSCs present a complex variety of regenerative behaviours at the clonal level, but the mechanisms underlying this diversity are still undetermined3–11. Recent advances in single cell RNA sequencing (scRNAseq) have revealed transcriptional differences amongst HSCs, providing a possible explanation for their functional heterogeneity12–17. However, the destructive nature of sequencing assays prevents simultaneous observation of stem cell state and function. To solve this challenge, we implemented expressible lentiviral barcoding, which enabled simultaneous analysis of lineages and transcriptomes from single adult HSCs and their clonal trajectories during long-term bone marrow reconstitution. Differential gene expression analysis between clones with distinct behaviour unveiled an intrinsic molecular signature that characterizes functional long-term repopulating HSCs. Probing this signature through in vivo CRISPR screening, we found the transcription factor Tcf15 to be required, and sufficient, to drive HSC quiescence and long-term self-renewal. In situ, Tcf15 expression labels the most primitive subset of true multipotent HSCs. In conclusion, our work elucidates clone-intrinsic molecular programs associated with functional stem cell heterogeneity, and identifies a mechanism for the maintenance of the self-renewing haematopoietic stem cell state.

The Trithorax-Group Protein ASH1L Regulates Hematopoietic Stem Cell Homeostasis Independently of Its Histone Methyltransferase Activity

Absent, small or homeotic discs 1-like (Ash1l) encodes the mammalian homolog of a Trithorax-group protein with a conserved SET domain that harbors histone H3 lysine 36 dimethyltransferase activity. Using mice with constitutive knockdown of Ash1l due to the insertion of a “gene trap” cassette in the first intron (Ash1lGT/GT mice), we previously reported that Ash1l is a critical regulator of quiescence and self-renewal in adult hematopoietic stem cells (HSCs). While wild-type HSCs could readily establish long-term bone marrow reconstitution after transplantation into irradiated recipients, Ash1l-deficient HSCs had markedly decreased quiescence and failed to establish long-term bone marrow reconstitution (Jones, Chase et al., JCI 2015). Here, we addressed three important questions to better understand the role of Ash1l in hematopoiesis: 1) What is the impact of complete, as opposed to partial, Ash1l loss on adult hematopoiesis? 2) Does Ash1l regulate HSC homeostasis in a cell-autonomous manner? 3) Is the catalytic activity of ASH1L required for its function?To move beyond the limitations of the constitutive knockdown Ash1lGT allele and address the first two questions, we studied newly generated mice carrying conditional knockout (cKO) Ash1l alleles (exon 13 flanked by loxP sites) along with the Mx1-Cre transgene. Induction of Mx1-Cre expression with poly(I:C) in Mx1-Cre+Ash1lf/f mice resulted in nonsense-mediated decay of Ash1l mRNA in hematopoietic cells, thereby completely knocking out Ash1l in adult hematopoietic stem and progenitor cells. Four weeks after inducing Ash1l inactivation in the hematopoietic compartment, we observed a profound depletion of HSCs and multipotent progenitors in Ash1l cKO mice similar to the phenotype of Ash1lGT/GTmice, indicating that conditional Ash1l knockout has a comparable impact on adult hematopoiesis to that of constitutive Ash1l knockdown. Of note, overt hematopoietic failure in steady-state conditions was not observed in either model. To address the second question, we transplanted a mixture of wild-type and Mx1-Cre+Ash1lf/f bone marrow into irradiated wild-type hosts, allowed donor bone marrow to occupy the wild-type niche and establish hematopoiesis, then induced Cre-mediated excision to inactivate Ash1l in Mx1-Cre+Ash1lf/f hematopoietic cells. Upon Cre induction, donor-derived Ash1lΔ/Δ HSCs and myeloid progeny were depleted or outcompeted by wild-type cells, consistent with the model that Ash1l regulates HSC homeostasis in a cell-autonomous manner. Given that Ash1l encodes a SET domain, we next sought to directly examine whether its catalytic activity is required for its role in regulating HSCs. We studied an Ash1l allele with an in-frame deletion of exon 11 and 12, resulting in preserved expression of ASH1L with internally deleted SET domain (ΔSET) (Miyazaki et al., PLOS Genetics 2013). Homozygous ΔSET mice were viable, and phenotypic analysis of adult ΔSET mice revealed normal frequencies of HSCs and multipotent progenitors, in contrast to our observations in Ash1lGT/GTand Ash1l cKO mice. Furthermore, transplanting ΔSET donor bone marrow into irradiated wild-type hosts resulted in sustained long-term reconstitution throughout primary, secondary and tertiary competitive transplantation assays, consistent with preserved HSC function and no alterations in cell cycle regulation. These findings establish that Ash1l regulates HSC homeostasis independently of its SET domain and histone methyltransferase activity. As ASH1L is a very large protein encoding multiple chromatin binding domains, we speculate that ASH1L may serve as a platform to recruit other partners to form novel protein complex(es) that regulate genes critical for HSC homeostasis. DisclosuresNo relevant conflicts of interest to declare.

Setd2 regulates quiescence and differentiation of adult hematopoietic stem cells by restricting RNA polymerase II elongation.

SET domain containing 2 (Setd2), encoding a histone methyltransferase, is associated with many hematopoietic diseases when mutated. By generating a novel exon 6 conditional knockout mouse model, we describe an essential role of Setd2 in maintaining the adult hematopoietic stem cells. Loss of Setd2 results in leukopenia, anemia, and increased platelets accompanied by hypocellularity, erythroid dysplasia, and mild fibrosis in bone marrow. Setd2 knockout mice show significantly decreased hematopoietic stem and progenitor cells except for erythroid progenitors. Setd2 knockout hematopoietic stem cells fail to establish long-term bone marrow reconstitution after transplantation because of the loss of quiescence, increased apoptosis, and reduced multiple-lineage terminal differentiation potential. Bioinformatic analysis revealed that the hematopoietic stem cells exit from quiescence and commit to differentiation, which lead to hematopoietic stem cell exhaustion. Mechanistically, we attribute an important Setd2 function in murine adult hematopoietic stem cells to the inhibition of the Nsd1/2/3 transcriptional complex, which recruits super elongation complex and controls RNA polymerase II elongation on a subset of target genes, including Myc. Our results reveal a critical role of Setd2 in regulating quiescence and differentiation of hematopoietic stem cells through restricting the NSDs/SEC mediated RNA polymerase II elongation.

Sox17-Mediated Maintenance of Fetal Intra-Aortic Hematopoietic Cell Clusters

During mouse development, definitive hematopoiesis is first detected around embryonic day 10.5 (E10.5) in the aorta-gonad-mesonephros (AGM) region, which exhibits intra-aortic cell clusters. These clusters are known to contain hematopoietic stem cells (HSCs). On the other hand, it is not clear how the cells in such clusters maintain their HSC phenotype and how they are triggered to differentiate. Here we found that an endodermal transcription factor marker, Sox17, and other F-group (SoxF) proteins, Sox7 and Sox18, were expressed in E10.5 intra-aortic cell clusters. Forced expression of any of these SoxF proteins, particularly Sox17, in E10.5 AGM CD45(low) c-Kit(high) cells, which are the major component of intra-aortic clusters, led to consistent formation of cell clusters in vitro during several passages of cocultures with stromal cells. Cluster-forming cells with constitutive Sox17 expression retained long-term bone marrow reconstitution activity in vivo. Notably, shutdown of exogenously introduced Sox17 gene expression resulted in immediate hematopoietic differentiation. These results indicate that SoxF proteins, especially Sox17, contribute to the maintenance of cell clusters containing HSCs in the midgestation AGM region. Furthermore, SoxF proteins play a pivotal role in controlling the HSC fate decision between indefinite self-renewal and differentiation during fetal hematopoiesis.

Enhanced Angpt1/Tie2 signaling affects the differentiation and long-term repopulation ability of hematopoietic stem cells

Angiopoietin-1 (Angpt1) signaling via the Tie2 receptor regulates vascular and hematopoietic systems. To investigate the role of Angpt1-Tie2 signaling in hematopoiesis, we prepared conditionally inducible transgenic (Tg) mice expressing a genetically engineered Angpt1, cartridge oligomeric matrix protein (COMP)-Angpt1. The effects of COMP-Angpt1 overexpression in osteoblasts on hematopoiesis were then investigated by crossing COMP-Angpt1 Tg mice with Col1a1-Cre Tg mice. Interestingly, peripheral blood analyses showed that 4 week (wk)-old (but not 8 wk-old) Col1a1-Cre+/COMP-Angpt1+ mice had a lower percentage of circulating B cells and a higher percentage of myeloid cells than Col1a1-Cre-/COMP-Angpt1+ (control) mice. Although there were no significant differences in the immunophenotypic hematopoietic stem and progenitor cell (HSPC) populations between Col1a1-Cre+/COMP-Angpt1+ and control mice, lineage(-)Sca-1(+)c-Kit(+) (LSK) cells isolated from 8 wk-old Col1a1-Cre+/COMP-Angpt1+ mice showed better long-term bone marrow reconstitution ability. These data indicate that Angpt1-Tie2 signaling affects the differentiation capacity of hematopoietic lineages during development and increases the stem cell activity of HSCs.

N-cadherin+ HSCs in fetal liver exhibit higher long-term bone marrow reconstitution activity than N-cadherin− HSCs

Adult hematopoietic stem cells (HSCs) are maintained in a microenvironment known as the stem cell niche. The regulation of HSCs in fetal liver (FL) and their niche, however, remains to be elucidated. In this study, we investigated the role of N-cadherin (N-cad) in the maintenance of HSCs during FL hematopoiesis. By using anti-N-cad antibodies (Abs) produced by our laboratory, we detected high N-cad expression in embryonic day 12.5 (E12.5) mouse FL HSCs, but not in E15.5 and E18.5 FL. Immunofluorescence staining revealed that N-cad+c-Kit+ and N-cad+ endothelial protein C receptor (EPCR)+ HSCs co-localized with Lyve-1+ sinusoidal endothelial cells (ECs) in E12.5 FL and that some of these cells also expressed N-cad. However, N-cad+ HSCs were also observed to detach from the perisinusoidal niche at E15.5 and E18.5, concomitant with a down-regulation of N-cad and an up-regulation of E-cadherin (E-cad) in hepatic cells. Moreover, EPCR+ long-term (LT)-HSCs were enriched in the N-cad+Lin−Sca-1+c-Kit+ (LSK) fraction in E12.5 FL, but not in E15.5 or E18.5 FL. In a long-term reconstitution (LTR) activity assay, higher engraftment associated with N-cad+ LSK cells versus N-cad− LSK cells in E12.5 FL when transplanted into lethally irradiated recipient mice. However, the higher engraftment of N-cad+ LSK cells decreased subsequently in E15.5 and E18.5 FL. It is possible that N-cad expression conferred higher LTR activity to HSCs by facilitating interactions with the perisinusoidal niche, especially at E12.5. The down-regulation of N-cad during FL hematopoiesis may help us better understand the regulation and mobility of HSCs before migration into BM.

Expression of Endothelial Protein C Receptor Confines Hematopoietic Stem Cell in Murine Fetal Liver.

Endothelial protein C receptor (EPCR or CD201) is expressed in the endothelial cells of large blood vessels, such as aorta, and contributes to the natural anticoagulant and anti-inflammatory system by activating protein C. Recent study showed that EPCR is also expressed in murine bone marrow, and that the majority of the EPCR-positive cells are the hematopoietic stem cells (HSC) in quiescent state, defined as the side population (sp) fraction in flow cytometry, suggesting the possibility of EPCR involvement in the hematopoiesis. In the murine mid-embryonic stage, on the other hand, the main location of hematopoiesis is fetal liver (FL), where the HSCs are highly proliferative. In this period of development, there are no quiescent HSCs, which is different from adult bone marrow. It is not clarified whether EPCR has any relevance to the fetal hematopoiesis. Here, we demonstrate that EPCR is involved in the hematopoiesis in FL. Immunostaining of murine FL (E12) clearly showed the EPCR-positive cells, and they were located adjacent or connected to the Lyve-1-positive sinusoidal endothelial cells. Some of those were endothelial-like, and others were in round shape, co-expressing c-Kit. In the HSC-enriched Lineage−Sca-1+c-Kit+ (LSK) fraction, the proportion of the EPCR-positive cells was approximately 30%. Sorted EPCR+/LSK and EPCR−/LSK cells were examined for the capability of bone marrow reconstitution and multilineage differentiation. In colony assay, the EPCR+/LSK cells exhibited higher proliferation and differentiation capability into the full range of the hematopoietic cells, including the megakaryocytes and erythroids, while most of the EPCR−/LSK-derived colonies were granulocytes and macrophages. Furthermore, the EPCR+/LSK and EPCR−/LSK cells isolated from donor FL were transplanted with the bone marrow-derived competitor cells into the lethally irradiated recipient mice, resulting in the striking difference between two fractions, i.e., robust long-term reconstitution of EPCR+/LSK cells and almost no reconstitution of EPCR−/LSK cells. The EPCR+/LSK cells were serially transplantable. These data collectively conclude that EPCR-expressing cells in LSK fraction of E12.5 FL exclusively exhibit the capability for long-term bone marrow reconstitution and self-renewal, as well as multilineage differentiation. Thus, the EPCR+/LSK fraction is the highly HSC-enriched population. The function of the endothelial molecule in HSCs will be discussed.

Hematopoietic stem cells expanded by fibroblast growth factor-1 are excellent targets for retrovirus-mediated gene delivery

For the study of the function of genes of interest in hematopoietic stem cells (HSCs) and for successful gene therapy, it is fundamental to develop a method of efficient gene transfer into HSCs. In mice experiments, efforts have been made to raise the transduction efficiency by modifying the vectors, administrating 5-fluorouracil (5-FU) to donor mice, selecting cytokine cocktails to better sustain the long-term repopulating potential of the stem cells, and so on. The objective of this study is to examine whether the use of fibroblast growth factor-1 (FGF-1)-expanded bone marrow cells provide an improved source for retroviral gene delivery to HSCs. Unfractionated bone marrow cells from one mouse were cultured in serum-free medium containing FGF-1. Both floating and attached cells were transferred to retronectin precoated dishes and infected with virus supernatant from MP34 cells stably transduced with pMY/GFP retrovirus. After 3-day infection, the green fluorescence protein-positive fraction was sorted and the cells were transplanted to lethally irradiated mice. The experiments illustrated that the number of bone marrow-derived competitive repopulation units (CRUs) was increased from 600 to 9300 per mouse after a 3-week culture period with FGF-1. Following retroviral transduction of the expanded cells, the absolute number of sorted retrovirus-transduced CRUs was 4200. Using these retrovirus-transduced cells in noncompetitive reconstitution assay, we achieved radiation protection and long-term bone marrow reconstitution in 100% of the recipients with average myeloid and lymphoid chimerisms of 70% and 50%, respectively, even if we transplanted 150 recipients with cells derived from a single donor mouse. In conclusion, FGF-1-expanded bone marrow cells constitute an excellent source of stem cells that could be used in a range of gene delivery protocols.

Gene correction in hematopoietic progenitor cells by homologous recombination.

Homologous recombination (gene targeting) has many desirable features for gene therapy, because it can precisely correct mutant genes and restore their normal expression, and random nonhomologous integration of DNA is infrequent in cells in which homologous recombination has occurred. There are, however, no reports of attempts to use homologous recombination to correct mutant genes in normal hematopoietic stem cells (HSCs), which are prime cells for therapy of a variety of hematological and other conditions, presumably because of their low abundance and uncertainty that homologous recombination can occur at a usable frequency in these cells. The experiments reported here encourage optimism in this respect by demonstrating targeted correction of a defective hypoxanthine phosphoribosyltransferase gene in hematopoietic progenitor cells that can form colonies in methylcellulose culture. These clonogenic cells are in the same lineage as HSCs but are more abundant and more mature and so less pluripotent. Corrected colonies were identified by their survival in selective medium after electroporation of correcting DNA into unfractionated mouse bone marrow cells and were confirmed by reverse transcription-PCR and sequencing. The observed frequency (4.4 +/- 3.3 x 10(-5) per treated clonogenic cell) is the same as in embryonic stem cells (2.3 +/- 0.4 x 10(-5)) with the same DNA and mutation. These data suggest that gene targeting to correct mutant genes eventually will prove feasible in HSCs capable of long-term bone marrow reconstitution.

Functional activity of murine CD34 +and CD34 − hematopoietic stem cell populations

The transmembrane glycoprotein CD34 is expressed on human hematopoietic stem cells and committed progenitors in the bone marrow, and CD34-positive selection currently is used to isolate bone marrow repopulating cells in clinical transplantation protocols. Recently, CD34 − hematopoietic stem cells were described in both humans and mice, and it was suggested that CD34 + murine bone marrow cells may lack long-term reconstituting ability. In this study, the long-term repopulating ability of CD34 + Lin − vs CD34 − Lin − cells was compared directly using syngeneic murine bone marrow transplantation. Highly purified populations of CD34 + Lin − and CD34 − Lin − cells each are able to reconstitute bone marrow, confirming that both populations contain hematopoietic stem cells; however, the number of hematopoietic stem cells in the CD34 + Lin − fraction is approximately 100-fold greater than the number in the CD34 − Lin − fraction. In competitive repopulation experiments, CD34 + stem cells are better able to engraft the bone marrow than are CD34 − cells. CD34 + Lin − cells provide both short- and long-term engraftment , but the CD34 − Lin − cells are capable of only long-term engraftment. Ex vivo, the CD34 + Lin − stem cells expand over 3 days in culture and maintain the ability to durably engraft animals in a serial transplant model. In contrast, when CD34 − Lin − cells are cultured using the same conditions ex vivo, the cell number decreases, and the cells do not retain the ability to repopulate the bone marrow. Thus, the CD34 + Lin − and CD34 − Lin − cells constitute two functionally distinct populations that are capable of long-term bone marrow reconstitution.

Role of bcl-2 in the Development of Lymphoid Cells From the Hematopoietic Stem Cell

AbstractTo investigate the role of bcl-2 in lymphohematopoiesis, a long-term bone marrow reconstitution system was established. Transplantation of 1,000 c-Kit+ Sca-1+ and lineage markers negative cells from bcl-2−/− mouse bone marrow resulted in long-term reconstitution of nonlymphoid cells. However, T cells were totally absent and B-lymphocyte development was severely impaired at a very early stage of differentiation in the chimeric mouse. On the other hand, transplantation of day 14 fetal liver cells from bcl-2−/− mice resulted in generation of both T and B cells in the recipient, albeit transiently. These data suggest that bcl-2 plays a critical role in the development of lymphoid progenitor cells from the hematopoietic stem cell (HSC), but is not essential for the development of nonlymphoid cells and the self-renewal of HSC. In addition, lymphopoiesis from fetal liver HSC appears to be less dependent on bcl-2 than adult bone marrow HSC.