Faculty Opinions recommendation of Clonal-level lineage commitment pathways of hematopoietic stem cells in vivo.

Author(s):  
Ivan Martin
Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 27-27
Author(s):  
Rong Lu ◽  
Agnieszka Czechowicz ◽  
Jun Seita ◽  
Irving L. Weissman

Abstract Abstract 27 Hematopoietic stem cells (HSCs) sustain the blood and immune systems through a complex differentiation process. This process involves several steps of lineage commitment and forms a paradigm for understanding cellular development, differentiation, and malignancy. While this step-wise differentiation has been extensively studied at the population level, little is known about the lineage commitment of individual HSC clones. The importance of understanding HSC differentiation at the clonal level has been raised by several recent studies suggesting that individual HSCs differentially contribute to various blood cell types and that the aggregate HSC differentiation at the population level is an amalgamation of the diverse lineage commitments of individual HSC clones. The distinct differentiation of individual HSCs may also be accentuated by their regulatory microenvironments, HSC niche. HSC niche may not affect all HSCs in an organism equally, and may instead act directly on resident HSC clones through direct contact or by tuning local cytokine concentrations. Knowledge of HSC clonal level lineage commitment will reveal new insights into HSC regulatory mechanisms and will improve our understanding of aging, immune deficiency, and many hematopoietic disorders involving an unbalanced hematopoietic system. Here, we provide a comprehensive map of in vivo HSC clonal development in mice. The clonal map was derived from the simultaneous tracking of hundreds of individual mouse HSCs in vivo using genetic barcodes. These unique barcodes were delivered into HSCs using a lentiviral vector to obtain a one-to-one mapping between barcodes and HSCs. Barcoded HSCs were then transplanted into recipient mice using standard procedures. Genetic barcodes from donor derived HSCs and their progenies were examined twenty-two weeks after transplantation using high-throughput sequencing. We found that the dominant differentiation of HSC clones is always present in pre-conditioned mice. In these recipients, a small fraction of engrafted HSCs become dominantly abundant at the intermediate progenitor stages, but not at the HSC stage. Thus, clonal dominance is a characteristic of HSC differentiation but not of HSC self-renewal. Additionally, the dominant differentiation of HSC clones exhibits distinct expansion patterns through various stages of hematopoiesis. We provide evidence that observed HSC lineage bias arises from dominant differentiation at distinct lineage commitment steps. In particular, myeloid bias arises from dominant differentiation at the first lineage commitment step from HSC to MPP, whereas lymphoid bias arises from dominant differentiation at the last lineage commitment step from CLP to B cells. We also show that dominant differentiation and lineage bias are interrelated and together delineate discrete HSC lineage commitment pathways. These pathways describe how individual HSC clones produce differential blood quantities and cell types. Multiple clonal differentiation pathways can coexist simultaneously in a single organism, and mutually compensate to sustain overall blood production. Thus, the distinct HSC differentiation characteristics uncovered by clonal analysis are not evident at the population level. We have also identified the lineage commitment profiles of HSC clones belonging to each pathway. These profiles elucidate the cellular proliferation and development of HSCs at the clonal level and demonstrate that distinct modes of HSC regulation exist in vivo. In summary, our in vivo clonal mapping reveals discrete clonal level HSC lineage commitment pathways. We have identified the cellular origins of clonal dominance and lineage bias, which may be the key hematopoietic stages where blood production and balance can be manipulated. These discoveries based on clonal level analysis are unexpected and unobtainable from conventional studies at the population level. Together, they open new avenues of research for studying hematopoiesis. Disclosures: No relevant conflicts of interest to declare.


2018 ◽  
Author(s):  
Rong Lu ◽  
Agnieszka Czechowicz ◽  
Jun Seita ◽  
Du Jiang ◽  
Irving L. Weissman

ABSTRACTWhile hematopoietic stem cells (HSCs) have been extensively studied at the population level, little is known about the lineage commitment of individual clones. Here, we provide comprehensive maps ofin vivoHSC clonal development in mice under homeostasis and after depletion of the endogenous hematopoietic system. Under homeostasis, all donor-derived HSC clones regenerate blood homogeneously throughout all measured stages and lineages of hematopoiesis. In contrast, after the hematopoietic system has been depleted by irradiation or by an anti-ckit antibody, only a small fraction of donor-derived HSC clones differentiates while dominantly expanding and exhibiting lineage bias. We identified the cellular origins of clonal dominance and lineage bias, and uncovered the lineage commitment pathways that lead HSC clones to differential blood production. This study reveals surprising alterations in HSC regulation by irradiation, and identifies the key hematopoiesis stages that may be manipulated to control blood production and balance.SIGNIFICANCE STATEMENTHematopoietic stem cells (HSCs) sustain daily blood production through a complex step-wise lineage commitment process. In this work, we present the first comprehensive study of HSC lineage commitment at the clonal level and identify new HSC regulatory mechanisms that are undetectable by conventional population level studies. First, we uncover distinct HSC clonal pathways that lead to differential blood production and imbalances. Second, we reveal that HSC regulation under physiological conditions is strikingly different from that after injury. Third, we present a comprehensive map of HSC activities in vivo at the clonal level.


2019 ◽  
Vol 116 (4) ◽  
pp. 1447-1456 ◽  
Author(s):  
Rong Lu ◽  
Agnieszka Czechowicz ◽  
Jun Seita ◽  
Du Jiang ◽  
Irving L. Weissman

While the aggregate differentiation of the hematopoietic stem cell (HSC) population has been extensively studied, little is known about the lineage commitment process of individual HSC clones. Here, we provide lineage commitment maps of HSC clones under homeostasis and after perturbations of the endogenous hematopoietic system. Under homeostasis, all donor-derived HSC clones regenerate blood homogeneously throughout all measured stages and lineages of hematopoiesis. In contrast, after the hematopoietic system has been perturbed by irradiation or by an antagonistic anti-ckit antibody, only a small fraction of donor-derived HSC clones differentiate. Some of these clones dominantly expand and exhibit lineage bias. We identified the cellular origins of clonal dominance and lineage bias and uncovered the lineage commitment pathways that lead HSC clones to different levels of self-renewal and blood production under various transplantation conditions. This study reveals surprising alterations in HSC fate decisions directed by conditioning and identifies the key hematopoiesis stages that may be manipulated to control blood production and balance.


Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3003-3003
Author(s):  
Shirong Li ◽  
Jing Fu ◽  
Jing Wu ◽  
Markus Y Mapara ◽  
Suzanne Lentzsch

Abstract Introduction: Previously we have shown that the immune modulatory drugs (IMiDs) downregulate GATA1 and PU.1 resulting in maturational arrest of granulocytes with accumulation of immature myeloid precursors and subsequent neutropenia. Our studies further revealed that similar to MM cells cereblon (CRBN) is critical for the mediation of the effects of IMiDS in hematopoietic stem cells (HSCs) and associated with decrease of IKZF1-dependent transcription factors such as GATA1 and PU.1, which are critical for development and maturation of neutrophils and erythrocytes as well as thrombocytes. Here we investigated the mechanism how IMIDs induce degradation of IKZF1 and confirmed our studies in vivo by using the humanized NOD/SCID/Gamma-c KO (NSG) mouse model. Methods and Results After we had shown that knockdown of CRBN in HCS mediates resistance to IMIDs (2014 ASH abstract 418) we assessed the impact of IKZF1 inhibition using two different approaches. First, we knocked down IKZF1 expression in CD34+ cells by shRNA lentivirus transduction. As expected, IKZF1 knockdown in CD34+ cells mimicked the effects of IMiDs resulting in increased CD34+ cell proliferation, CD33+ cell expansion (flow cytometry) and shift of lineage commitment from BFU-E to CFU-G (colony assay). Knockdown of IKZF1 was associated with decreased GATA1 and PU.1 expression at both mRNA and protein levels. Next, we generated a mutant IKZF1 by substituting Glutamine Q146 to Histidine, which abrogates IKZF1 ubiquitination induced by CRBN. CD34+ cells were transduced with lentiviral constructs to overexpress IKZF1-WT or IKZF1-Q146H. POM failed to induce IKZF1 degradation in IKZF1-Q146H-OE CD34+ cells, indicating CRBN binding to IKZF1 and subsequent ubiquitination is critical in this process. Functional assays further confirmed that IKZF1-Q146H CD34+ cells were resistant to POM induced CD33+ cell expansion and shift in lineage commitment from BFU-E to CFU-G. Since conventional mouse models are not applicable to test IMIDs in vivo due to the fact that IMIDs do not bind to mouse CRBN (Kronke, Fink et al. 2015), we established a humanized mouse model resembling human hematopoiesis. In this model, NOD/SCID/Gamma-c KO (NSG) mice received human fetal thymus grafts and 105 CD34+ fetal liver cells to generate human hematopoiesis including functional T-cells. After establishing human hematopoiesis mice were injected with POM (0.3 mg/kg) i.v every 2 days for 3 weeks. Analysis of bone marrow revealed that POM treatment significantly induced granulocyte/macrophage progenitor cells (CD34+ CD38+ CD45RA+ cells) at the expense of common lymphoid progenitors (CD34+ CD10+ cells). The shift into myelopoiesis is consistent with our in vitro finding that IMiDs affect lineage commitment. Conclusion: In summary, our results demonstrate that IMiDs affect CD34+ cell fate via CRBN and IKZF1 mediated mechanism. These results will be helpful to elucidate the mechanism of IMiDs on lineage commitment and maturation in HSCs. Also establishment of the humanized xenograft mice model may provide an advanced platform for the analysis of human hematopoiesis and human immune responses to IMiDs as well development of secondary hematologic malignancies in vivo. Disclosures Lentzsch: Axiom: Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.


Author(s):  
Fatima Aerts-Kaya

: 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.


1987 ◽  
Vol 5 (3) ◽  
pp. 231-241 ◽  
Author(s):  
Vincent S. Gallicchio ◽  
Thomas D. Watts ◽  
George P. Casale ◽  
Philip M. Bartholomew

1993 ◽  
Vol 90 (8) ◽  
pp. 3760-3764 ◽  
Author(s):  
W. H. Fleming ◽  
E. J. Alpern ◽  
N. Uchida ◽  
K. Ikuta ◽  
I. L. Weissman

Blood ◽  
2006 ◽  
Vol 108 (4) ◽  
pp. 1189-1197 ◽  
Author(s):  
Hua Tang ◽  
Zhenhong Guo ◽  
Minghui Zhang ◽  
Jianli Wang ◽  
Guoyou Chen ◽  
...  

Abstract Regulatory dendritic cells (DCs) have been reported recently, but their origin is poorly understood. Our previous study demonstrated that splenic stroma can drive mature DCs to proliferate and differentiate into regulatory DCs, and their natural counterpart with similar regulatory function in normal spleens has been identified. Considering that the spleen microenvironment supports hematopoiesis and that hematopoietic stem cells (HSCs) are found in spleens of adult mice, we wondered whether splenic microenvironment could differentiate HSCs into regulatory DCs. In this report, we demonstrate that endothelial splenic stroma induce HSCs to differentiate into a distinct regulatory DC subset with high expression of CD11b but low expression of Ia. CD11bhiIalo DCs secreting high levels of TGF-β, IL-10, and NO can suppress T-cell proliferation both in vitro and in vivo. Furthermore, CD11bhiIalo DCs have the ability to potently suppress allo-DTH in vivo, indicating their preventive or therapeutic perspectives for some immunologic disorders. The inhibitory function of CD11bhiIalo DCs is mediated through NO but not through induction of regulatory T (Treg) cells or T-cell anergy. IL-10, which is secreted by endothelial splenic stroma, plays a critical role in the differentiation of the regulatory CD11bhiIalo DCs from HSCs. These results suggest that splenic microenvironment may physiologically induce regulatory DC differentiation in situ.


Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2309-2309
Author(s):  
Jian Huang ◽  
Peter S. Klein

Abstract Abstract 2309 Hematopoietic stem cells (HSCs) maintain the ability to self-renew and to differentiate into all lineages of the blood. The signaling pathways regulating hematopoietic stem cell (HSCs) self-renewal and differentiation are not well understood. We are very interested in understanding the roles of glycogen synthase kinase-3 (Gsk3) and the signaling pathways regulated by Gsk3 in HSCs. In our previous study (Journal of Clinical Investigation, December 2009) using loss of function approaches (inhibitors, RNAi, and knockout) in mice, we found that Gsk3 plays a pivotal role in controlling the decision between self-renewal and differentiation of HSCs. Disruption of Gsk3 in bone marrow transiently expands HSCs in a b-catenin dependent manner, consistent with a role for Wnt signaling. However, in long-term repopulation assays, disruption of Gsk3 progressively depletes HSCs through activation of mTOR. This long-term HSC depletion is prevented by mTOR inhibition and exacerbated by b-catenin knockout. Thus GSK3 regulates both Wnt and mTOR signaling in HSCs, with opposing effects on HSC self-renewal such that inhibition of Gsk3 in the presence of rapamycin expands the HSC pool in vivo. In the current study, we found that suppression of the mammalian target of rapamycin (mTOR) pathway, an established nutrient sensor, combined with activation of canonical Wnt/ß-catenin signaling, allows the ex vivo maintenance of human and mouse long-term HSCs under cytokine-free conditions. We also show that combining two clinically approved medications that activate Wnt/ß-catenin signaling and inhibit mTOR increases the number of long-term HSCs in vivo. Disclosures: No relevant conflicts of interest to declare.


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