Combined Single Cell Lineage and Transcriptome Sequencing Unveils Cell-Autonomous Regulators of Hematopoietic Stem Cell Fate

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 446-446
Author(s):  
Alejo E Rodriguez-Fraticelli ◽  
Caleb S Weinreb ◽  
Allon Moshe Klein ◽  
Shou-Wen Wang ◽  
Fernando D Camargo
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Conflicts Of Interest ◽  
Large Fraction ◽  
Cell Lineage ◽  
Gene Signature ◽  
Hematopoietic Stem ◽  
Cell State

Blood regeneration upon transplantation relies on the activity of long-term repopulating hematopoietic stem cells (LT-HSCs). One of the major controversies in hematopoiesis relates to the apparently different properties that HSCs have in transplantation versus unperturbed settings. In unperturbed steady state hematopoiesis, the most potent HSCs appear to be mostly dormant, and only producing platelet-lineage cells. In turn, upon transplant, even a single transplanted HSC can actively divide and regenerate hundreds of millions of blood progenitors of all lineages. It would thus appear that HSCs have different fundamental properties in each study system. However, most transplantation studies have only tracked the lineage output of the transplanted HSC clones, and rarely the regeneration of the HSC compartment itself. In addition, clonal assays have not been performed at sufficient resolution to fully capture the diversity and clonal complexity of the regenerated HSC compartment. Here, we have used expressible barcodes, which can be sequenced in conventional single cell RNAseq assays, to simultaneously record the functional outcomes and transcriptional states of thousands of HSCs. Our analysis revealed multiple clonal HSC behaviors following transplantation that drastically differ in their differentiation activity, lineage-bias and self-renewal. Surprisingly, we witnessed a large fraction of clones that efficiently repopulate the HSC compartment but show limited contribution to differentiated progeny. Furthermore, these inactive clones have increased competitive multilineage serial repopulating capacity, implying that shortly after transplant a subset of clones reestablishes the native-like LT-HSC behaviors. Our results also argue that this clonal distribution of labor is controlled by cell autonomous, heritable properties (i.e. the epigenetic cell state). Then, using only our clonal readouts to segregate single HSC transcriptomes, we unveiled the transcriptional signatures that associated with unique HSC outcomes (platelet bias, clonal expansion, dormancy, etc.) and unraveled, for the first time, a gene signature for functional long-term serially repopulating clones. We interrogated the drivers of this cell state using an in vivo inducible CRISPR screening and identified 5 novel regulators that are required to regenerate the HSC compartment in a cell autonomous fashion. In conclusion, we demonstrate that functional LT-HSCs share more similar properties in native and transplantation hematopoiesis than previously expected. Consequently, we unveil a definition of the essential, common functional properties of HSCs and the molecular programs that control them. Figure 1 Disclosures No relevant conflicts of interest to declare.

Pax5 determines B- versus T-cell fate and does not block early myeloid-lineage development

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4342-4346 ◽  
Author(s):  
Claudiu V. Cotta ◽  
Zheng Zhang ◽  
Hyung-Gyoon Kim ◽  
Christopher A. Klug
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Fate ◽  
Nk Cell ◽  
Cell Lineage ◽  
Blood Analysis ◽  
Hematopoietic Stem ◽  
Peripheral Blood Analysis

Abstract Progenitor B cells deficient in Pax5 are developmentally multipotent, suggesting that Pax5 is necessary to maintain commitment to the B-cell lineage. Commitment may be mediated, in part, by Pax5 repression of myeloid-specific genes. To determine whether Pax5 expression in multipotential cells is sufficient to restrict development to the B-cell lineage in vivo, we enforced expression of Pax5 in hematopoietic stem cells using a retroviral vector. Peripheral blood analysis of all animals reconstituted with Pax5-expressing cells indicated that more than 90% of Pax5-expressing cells were B220+ mature B cells that were not malignant. Further analysis showed that Pax5 completely blocked T-lineage development in the thymus but did not inhibit myelopoiesis or natural killer (NK) cell development in bone marrow. These results implicate Pax5 as a critical regulator of B- versus T-cell developmental fate and suggest that Pax5 may promote commitment to the B-cell lineage by mechanisms that are independent of myeloid gene repression.

T(15;17)-PML/RAR-Induced Leukemogenesis: Long-Term Repopulating Hematopoietic Stem Cells as the Initial Target and More Mature Progenitors as the Potential Targets for Final Leukemic Transformation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3980-3980 ◽  
Author(s):  
Claudia Oancea ◽  
Brigitte Rüster ◽  
Jessica Roos ◽  
Afsar Ali Mian ◽  
Tatjana Micheilis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Population ◽  
Conflicts Of Interest ◽  
Cell Model ◽  
Leukemic Cell ◽  
Hematopoietic Stem ◽  
Proliferation Potential

Abstract Abstract 3980 Poster Board III-916 Stem cells have been shown to play an important role in the pathogenesis and maintenance of a significant number of malignancies, including leukemias. Similar to normal hematopoiesis the AML cell population is thought to be hierarchically organized. According to this model, only a few stem cells (LSC) are able to initiate and maintain the disease. The inefficient targeting of the leukemic stem cells (LSC) is considered responsible for relapse after the induction of complete hematologic remission (CR) in AML. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by the t(15;17) translocation and expression of the PML/RARα fusion protein. Treatment of APL with all-trans retinoic acid (t-RA) as monotherapy induces CR, but not molecular remission (CMR), followed by relapse within a few months. In contrast arsenic as monotherapy induces high rates of CR and CMR followed by a long relapse-free survival. We recently have shown that in contrast to t-RA, arsenic efficiently targets PML/RAR-positive stem cells, whereas t-RA increases their proliferation. For a better characterization of LSC in APL which has to be targeted for an efficient eradication of the disease we wanted to characterize the leukemia-initiating cell and the cell population able to maintain the disease in vivo. The model was based on a classical transduction/transplantation system of murine Sca1+/lin- HSC combined with a novel approach for the enrichment of transformed cells with long-term stem cell properties. We found that PML/RAR induced leukemia from the Sca1+/lin- HSC with a frequency of 40% and a long latency of 8-12 months independently of its capacity to increase dramatically replating efficiency and CFU-S12 potential as expression of the differentiation block and proliferation potential of derived committed progenitors. Based on the hypothesis that PML/RAR exerts its leukemogenic effects on only a small proportion of the Sca1+1/lin- population, we proceeded to select and to amplify rare PML/RAR-positive cells with the leukemia-initiating potential, by a negative selection of cell populations with proliferation potential without long term stem cell-capacity (LT). Therefore we expressed PML/RAR in Sca1+/lin- cells and enriched this population for LT- (lin-/Sca1+/c-Kit+/Flk2-) and ST-HSC (lin-/Sca1+/c-Kit+/Flk2+). After a passage first in semi-solid medium for 7 days and subsequent transplantation into lethally irradiated mice, cells from the ensuing CFU-S day12 were again transplanted into sublethally recipient mice. After 12 to 36 weeks, 6/6 mice developed acute myeloid leukemia without signs of differentiation in the group transplanted with the lin-/Sca1+/c-Kit+/Flk2- population but not from that transplanted with lin-/Sca1+/c-Kit+/Flk2+ cells. This leukemia was efficiently transplanted into secondary recipients. The primary leukemic cell population gave origin to 6 clearly distinct subpopulations defined by surface marker pattern as an expression of populations with distinct differentiation status, able - after sorting - to give leukemia in sublethally irradiated recipients: Sca1+/c-Kit+/CD34- (LT-HSC), Sca1+/c-Kit+/CD34+ (ST-HSC), Sca1-/c-Kit+, B220lo/GR1+/Mac1+, B220hi/GR1+/Mac1+, B220-/Gr1-/Mac1-. Interestingly, all leukemias from the different population presented an identical phenotype. These findings strongly suggest that there is a difference between a leukemia-initiating (L-IC) and leukemia-maintaining (L-MC) cell population in the murine PML/RAR leukemia model. In contrast to the L-IC, represented by a very rare subpopulation of primitive HSC, recalling a hierarchical stem cell model, the L-MC is represented by a larger cell population with a certain grade of phenotypical heterogeneity, but a high grade of functional homogeneity recalling a stochastic cancer induction model. Disclosures: No relevant conflicts of interest to declare.

In Vivo Selection Unmasks a Dormant Pool of Repopulating Hematopoietic Clones

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 242-242
Author(s):  
Jennifer E Adair ◽  
Lauren E Schefter ◽  
Daniel R Humphrys ◽  
Kevin G Haworth ◽  
Jonah D Hocum ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Selective Advantage ◽  
First Year ◽  
Short Term ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Chemotherapy Administration ◽  
Cd34 Cells

Abstract Long-term clonal tracking studies utilizing hematopoietic stem and progenitor cells (HSPCs) in nonhuman primates receiving myeloablative transplantation demonstrate a successive pattern of repopulation: short-term repopulating cells are succeeded by long-term clones. However, the duration of short-term repopulation and the numbers of clones contributing to either short or long-term repopulation are unclear. Here, we tracked >11,000 unique clones in 8 pigtail macaques for up to 9 years following myeloablative transplantation with autologous, lentivirus gene-modified CD34+ HSPCs. Seven of these animals received cells expressing the P140K mutant methylguanine methyltransferase transgene, which is resistant to the combination of O6-benzylguanine (O6BG) and bis-chloroethylnitrosourea (BCNU) chemotherapy, thus conferring a selective advantage to gene-modified cells in vivo. After transplantation and before in vivo selection with O6BG/BCNU, we observed a successive pattern of hematopoietic reconstitution, with short-term clones declining within 100 days after transplantation. Within the first year after transplant, the percent of persistent clones varied from animal-to-animal, ranging from 8% to 54% of clones detected at a >1% frequency, and remained stable in the absence of selective pressure. Importantly, when animals engrafted with P140K-expressing cells were administered O6BG/BCNU we observed novel clonal patterns, which directly correlated with transplanted cell dose and time of chemotherapy administration after transplant. In all animals, chemotherapy induced emergence of previously undetected clones. In animals receiving ≤12x106 CD34+ cells/kg at the time of transplant (n = 4), chemotherapy also induced a re-emergence of previously declined short-term repopulating clones or a stabilization (i.e. decreased fluctuation) of repopulating clones identified between 100 days and 1 year after transplant. However, in animals receiving robust cell doses, ≥35x106 CD34+ cells/kg (n = 2), chemotherapy more than 1 year after transplant induced a completely novel clonal repertoire. In one animal receiving 22x106 CD34+ cells/kg at transplant, chemotherapy administration beginning <1 year (253 days) after transplant induced clonal stability, which was maintained through two additional chemotherapy treatments. These data suggest that some short-term repopulating clones may have long-term repopulation ability, but revert to a dormant phase within the first year after transplant. Additionally, these data indicate that transplant of excess repopulating cells results in early dormancy of a large proportion of repopulating clones. Together, these findings suggest that previous estimates of HSPC frequency based on clone tracking are an underestimate of true graft repopulation potential. Disclosures No relevant conflicts of interest to declare.

Leptin Receptor As a Marker for a Subset of Long-Term Functional Hematopoietic Stem Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-32
Author(s):  
Thao Trinh ◽  
James Ropa ◽  
Arafat Aljoufi ◽  
Scott Cooper ◽  
Edward F. Srour ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Leptin Receptor ◽  
Conflicts Of Interest ◽  
Gene Set Enrichment Analysis ◽  
Type I ◽  
Hematopoietic Stem ◽  
Therapeutic Implications

The hematopoietic system is maintained by the hematopoetic stem and progenitor cells (HSCs/HPCs), a group of rare cells that reside in a hypoxic bone marrow (BM) microenvironment. Leptin (Lep) is well-known for its neuroendocrine and immunological functions, and its receptor (Lepr) has been studied extensively in the BM niche cells. Yet, its biological implications in HSC/HPC biology remained largely unknown. In this study, we hypothesized that Lepr-expressing HSCs/HPCs are functionally and transcriptomically distinct from their negative counterparts. To test our hypothesis, we utilized both in vitro and in vivo approaches. We first employed Fluorescence-activated cell sorting (FACS) analysis to confirm expression of Lepr on HSCs/HPCs in adult mouse BM. We then isolated equal numbers of Lepr+Lineage-Sca1+cKit+ (LSK cells - a heterogenous population of long-term, short-term HSCs and multipotent HPCs) and Lepr-LSK cells from C57BL/6 (CD45.2+) mouse BM to perform colony-forming unit (CFU) assay and competitive transplantation assay, which also included using competitor cells from BoyJ (CD45.1+) unseparated BM and lethally-irradiated F1 (CD45.1+CD45.2+) as hosts. To determine whether Lepr can further hierarchize HSCs into two distinct populations, we repeated the competitive transplants using freshly isolated C57BL/6 Lepr+HSCs or Lepr-HSCs cells instead. At the end of primary transplants, whole BM were analyzed for donor chimerisms in the peripheral blood (PB) and BM as well as transplanted in a non-competitive fashion into lethally-irradiated secondary recipients. To gain mechanistic insights, we assessed homing potential as homing plays a role in increased engraftment. We also performed bulk RNA-seq using freshly sorted BM Lepr+HSCs or Lepr-HSCs to elucidate potential molecular pathways that are responsible for the differences in their functional capacity. By phenotypic studies, our FACS analyses showed that Lepr+ cells represented a smaller population within the hematopoietic compartment in the BM. However, HSCs contained a higher percentage of Lepr+ cells than other HPC populations. By functional assessments, Lepr+LSK cells were more highly enriched for colony-forming progenitor cells in CFU assay as compared to Lepr-LSK cells. Interestingly, Lepr+LSK cells exhibited more robust engraftment capability in primary transplants and substantial self-renewal capacity in secondary transplants throughout different time points in both PB and BM. In addition, Lepr+HSCs showed significantly higher donor chimerisms in PB month 1, 2, 4 and BM month 4 with similar lineage output compared to Lepr-HSCs. Higher engraftment could be due to increased homing of HSCs to the BM; however, Lepr+HSCs and Lepr-HSCs showed similar homing capacity as well as levels of surface CXCR4 expression. Molecularly, Fast Preranked Gene Set Enrichment Analysis (FGSEA) showed that Lepr+HSCs were enriched for Type-I Interferon and Interferon-gamma response pathways with Normalized Enrichment Scores of 2 or higher. Lepr+HSC transcriptomic study also revealed that these cells as compared to Lepr-HSCs expressed significantly higher levels of genes involved in megakaryopoiesis and proinflammatory immune responses including the NF-κB subunits (Rel and Relb). Interestingly, both IFN-γ and NF-κB signalings have been demonstrated to be critical for the emergence of HSCs from the hemogentic endothelium during embryonic development. In summary, although Lepr+LSK cells occupied a minor fraction compared to their negative counterparts in the BM, they possessed higher colony-forming capacity and were more highly enriched for long-term functional HSCs. In line with this, Lepr+HSCs engrafted significantly higher and self-renewed more extensively than Lepr-HSCs, suggesting that Lepr not only can be used as a marker for functional HSCs but also further differentiate HSCs into two functionally distinguishable populations. Intriguingly, Lepr+HSCs were characterized with a proinflammatory transcriptomic profile that was previously suggested to be critical for the development of HSCs in the embryo. All together, our work demonstrated that Lepr+HSCs represent a subset of highly engrafting adult BM HSCs with an embryonic-like transcriptomic signature. This can have potential therapeutic implications in the field of hematopoietic transplantation as Lepr is highly conserved between mice and human. Disclosures No relevant conflicts of interest to declare.

Myosin-II Is a Major Modulator of Human Hematopoietic Stem Cell Proliferation and Differentiation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2343-2343
Author(s):  
Jae-Won Shin ◽  
Amnon Buxboim ◽  
Dennis E Discher
Keyword(s):  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myosin Ii ◽  
Dependent Manner ◽  
Specific Flow ◽  
Hematopoietic Stem ◽  
Matrix Elasticity ◽  
Proliferation And Differentiation

Abstract Abstract 2343 Non-muscle myosin-II (NMM-II) promotes cell division, membrane rigidity and adhesion to a rigid matrix, and so NMM-II activity might be predicted to be low in dormant hematopoietic stem cells (HSCs) and to increase with differentiation. Deletion of NMM-II is known to be embryonic lethal, but its role in adult HSC differentiation is not known. Recently, we showed that sustained pharmacological inhibition of NMM-II together with soft 2D matrices like the perivascular niches in marrow, rather than rigid like bone, maximizes both MK maturation and platelet generation (Shin et al., PNAS, 2011; 108:11458-63). HSCs exhibit some similarities to mature MKs in that long-term HSCs remain undivided in vivo while various progenitors and maturing cells rapidly expand in number. Here, reversible inhibition of NMM-II sustained over several cell cycles enriches long-term HSCs up to 20 fold by selective elimination of proliferating progenitors. CFSE dilution analysis indicates that inhibition of NMM-II eliminates the accumulation phase of hematopoietic progenitors and accelerates natural cell death rate by apoptosis. Interestingly, supplementation of G-CSF significantly enhances HSC survival under NMM-II inhibition and further accelerates progenitor elimination. Molecular profiling and functional analyses indicate that NMM-II isoforms play distinct roles during HSC differentiation. NMM-IIA is a marker for differentiation with significantly lower expression in HSCs than committed progenitors, which is consistent with greater membrane flexibility of HSCs measured by micropipette aspiration. In contrast, NMM-IIB is 5 fold higher in HSCs and progenitors than differentiated CD34− cells. HSC and progenitor numbers are also sensitive to matrix elasticity in a NMM-II dependent manner, with maximal expansion on soft and high-density fibronectin matrices (not collagen). However, upon NMM-II inhibition, the extent of HSC enrichment relative to multipotent progenitors is more sensitive to matrix ligand density than matrix elasticity. To identify physiological mechanisms of regulating NMM-II activity during early HSC differentiation, we investigated post-translational modifications of NMM-IIA, specifically the de-activating and isoform-specific phosphorylation at myosin Ser1943 (pS1943) in HSC and progenitors. In a phospho-specific flow cytometry approach, pS1943 level proves highest in HSCs and decreases during differentiation with Tpo and G-CSF but not SCF alone. TGF-beta inhibits the reduction of pS1943 level, consistent with TGF-beta's known role in HSC hibernation. Therefore, pS1943 level dictates HSC enrichment and parallels the dose-response to pharmacological NMM-II inhibitors. Furthermore, phospho-mimetic mutation of NMM-IIA at Ser1943 decreases cytoskeletal integrity, increases membrane flexibility, and limits matrix elasticity sensing, indicating that biophysical properties of HSCs can also be regulated by HSC-specific signaling via NMM-IIA heavy chain phosphorylation. Myosin-inhibited CD34+-derived bone marrow cells show reduced colony-forming unit progenitors in vitro, but maintain functional long-term HSCs in vivo in the marrows of xenografted mice with an added benefit to increase platelet circulation simultaneously. Therefore, myosin-II inhibition and soft, high ligand fibronectin constitutes an important ‘microenvironment mimetic’ approach to enrichment of long-term HSCs. Myosin-II is clearly a central, matrix-regulated node for HSC proliferation and differentiation. Disclosures: No relevant conflicts of interest to declare.

Single Cell Proteomics Reveals Novel Cytokine-Producing Function of Hematopoietic Stem and Progenitor Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 26-26
Author(s):  
Jimmy L. Zhao ◽  
Chao Ma ◽  
Ryan O'Connell ◽  
Dinesh S. Rao ◽  
James Heath ◽  
...  
Keyword(s):  
Stem Cells ◽  
Immune Response ◽  
Single Cell ◽  
Progenitor Cells ◽  
Cytokine Production ◽  
Conflicts Of Interest ◽  
Severe Neutropenia ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Abstract 26 During infection, hematopoietic stem and progenitor cells (HSPCs) are called upon to proliferate and differentiate to produce more innate and adaptive immune cells to combat infection. Traditionally, HSPCs are thought to respond to depletion of downstream hematopoietic cells during infection. More recent evidence suggests that HSPCs may respond directly to infection and pro-inflammatory cytokines. However, little is known about the direct immune response of HSPCs and the molecular signaling regulating this response upon sensing an infection. In this study, we have combined transgenic and genetic knockout mouse models with a novel single cell barcode proteomics microchip technology to tackle these questions. We show that although long-term hematopoietic stem cells (HSCs) (defined by Lineage-cKit+Sca1+CD150+CD48-) do not secrete cytokines upon toll-like receptor (TLR) stimulation, short-term HSCs and multipotent progenitor cells (MPPs) (defined by Lineage-cKit+Sca1+, referred to as LKS thereafter) can produce copious amounts of cytokines upon direct TLR-4 and TLR-2 stimulation, indicating that LKS cells can directly participate in an immune response by producing a myriad of cytokines, upon a bacterial infection. Within the population of LKS cells we detect multiple functional subsets of cells, specialized in producing myeloid-like, lymphoid-like or both types of cytokines. Moreover, we show that the cytokine production by LKS cells is regulated by the NF-κB activity, as p50-deficient LKS cells show reduced cytokine production while microRNA-146a (miR-146a)-deficient LKS cells show significantly increased cytokine production. As long-term HSCs differentiate, they start to gain effector immune function much earlier than we had originally anticipated. In light of this finding, we should start to view the stepwise differentiation scheme of HSCs, and perhaps all other stem cells, as a strategy to sequentially gain functional capacity, instead of simply losing stemness and self-renewal ability. The remarkable ability of LKS cells to produce copious amounts of cytokines in response to bacteria may provide some protective immunity during severe neutropenia and lymphopenia or in the early stage of HSC transplantation. This study further extends the functions of NF-κB to include the regulation of primitive hematopoietic stem and progenitor cells and provides direct evidence of the bacteria-responding ability of HSPCs through the TLR/NF-κB axis. The single cell barcode proteomics technology can be widely applied to study proteomics of other rare cells or heterogeneous cell population at a single cell level. Disclosures: No relevant conflicts of interest to declare.

Thrombopoietin-Receptor Signalling Induces Proliferation of Dormant HSC.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2343-2343
Author(s):  
Larisa V. Kovtonyuk ◽  
Markus G. Manz ◽  
Hitoshi Takizawa
Keyword(s):  
Conflicts Of Interest ◽  
Cxcr4 Antagonist ◽  
Extrinsic Factors ◽  
Subsequent Increase ◽  
Hematopoietic Stem ◽  
Regulatory Pathways ◽  
Surface Receptors ◽  
Early Results

Abstract Abstract 2343 Lifelong blood production is maintained by a very rare population of self-renewing hematopoietic stem cells (HSCs) in bone marrow (BM). Proliferation, differentiation and survival of HSC toward stepwise hematopoietic cell development needs to be tightly controlled by cell intrinsic and extrinsic factors, as excess or insufficient production of mature blood cells potentially leads to neoplasia or aplasia. HSCs and progenitors (HSPCs) are equipped with cell surface receptors for different cytokines or chemokines (Kaushansky, NEJM 2006), and thus can integrate external signals, leading finally to proliferation and subsequent increase of hematopoietic cells in demand. Some of these regulatory pathways are already exploited in clinical settings: CXCR4 antagonists for HSPC mobilization, human granulocyte colony-stimulating factor (hG-CSF) for HSC mobilization and myeloid regeneration, and thrombopoietin agonists (THPO) for improving thrombocytopenia. However, despite their clinical use, little is known about the influence of these molecules on HSC. We established in vivo HSC divisional tracking with CFSE (5(6)-carboxyfluorescein diacetate N-succinimidyl ester). This allows to track single HSC division with high resolution, and subsequently to test biological activity of HSC-containing fractions (LKS) with different divisional history (Takizawa et al., JEM 2011). Using this system we evaluated the effects of systemic administration of human fms-related tyrosine kinase 3 ligand (hFlt3L), hG-CSF (Filgrastim), CXCR4 antagonist (AMD3100), and the THPO receptor (cMpl) agonist (Romiplostim) on HSC division. CFSE-labeled LKS were transferred into non-irradiated steady-state recipients. The non-dividing cell fraction was defined by the CFSE profile of CD4+CD62L+ T cells. One week after transplantation mice were injected with PBS or respective reagents daily or every other day for over one week. Three weeks after transfer, phenotypic BM analysis demonstrated that most of donor LKS had undergone several divisions while a small fraction of LKS remained undivided in PBS treated control mice (Figure 1a), containing long term self-renewing HSCs with at least 20–30% frequency (Takizawa et al., JEM 2011). Administration of hFlt3L increased the percentage of intermediate (1–5x divided) and fast cycling (>5x divided) LKS, which mainly contains CD150- Flt3+ multipotent progenitor cells (Figure 1b). Upon injections of cMpl agonist all donor LKS divided more than once, leaving no cells in quiescent fraction, with substantial expansion of CD150+ cells in the divided fraction. CXCR4 antagonist and hG-CSF administration had little effect on LKS proliferation. These data suggest that cMpl agonist drives dormant cells into proliferation, whereas hG-CSF has little effect on LKS division. To determine whether cMpl agonist increases the turnover of functionally defined, bona fide HSCs, we performed secondary transplantation of 0–1, 2–4, and ≥5x divided LKS. Twenty fast- (≥5x divided cells at 3 weeks), slow-cycling (2–4x divided) or relatively dormant LKS Flt3- cMpl+ cells (0–1x divided) were sorted from mice treated with PBS or cMpl agonist, and transplanted into lethally irradiated mice. Early results demonstrate increased percentage of secondary recipient engrafted with 2–4x divided cells from primary animals treated with cMpl agonist compared to those cells from PBS treated control Our results thus suggest that cMpl agonists have mitogenic activity not only for megakaryocyte progenitors but also for HSCs. How far this holds true in the human species needs to be determined. However, it should be taken in consideration given clinical data on evolution of pre-existing clonal myeloid diseases under cMpl agonist treatment (Dantoni, ASH abstract 2011), and also when treatment is applied long-term to patients with primary non-clonal hematopoietic diseases as immune thrombocytopenia or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Integrated single-cell transcriptomic and epigenetic analyses of cell-state transition and lineage commitment in the embryonic mouse cerebellum

2021 ◽  
Author(s):  
Khouri Farah-Nagham ◽  
Qiuxia Guo ◽  
Kerry Morgan ◽  
Jihye Shin ◽  
James Y.H. Li
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Regulatory Networks ◽  
State Transition ◽  
Control Cell ◽  
Cellular Heterogeneity ◽  
Embryonic Mouse ◽  
Molecular Control ◽  
Cell State

Recent studies using single-cell RNA-seq have revealed cellular heterogeneity in the developing mammalian cerebellum, yet the regulatory logic underlying this cellular diversity remains to be elucidated. Using integrated single-cell RNA and ATAC analyses, we resolved developmental trajectories of cerebellar progenitors and identified putative trans- and cis-elements that control cell state transition. We reverse-engineered gene regulatory networks (GRNs) of each cerebellar cell type. Through in silico simulations and in vivo experiments, we validated the efficacy of GRN analyses and uncovered the molecular control of a newly identified stem zone, the posterior transitory zone (PTZ), which contains multipotent progenitors for granule neurons, Bergmann glia, and choroid plexus epithelium. Importantly, we showed that perturbing cell fate specification of PTZ progenitors causes posterior cerebellar vermis hypoplasia, the most common cerebellar birth defect in humans. Our study provides a foundation for comprehensive studies of developmental programs of the mammalian cerebellum.

scholarly journals Long-Term Hematopoietic Clonal Stability Tracked Using Molecular Barcoding in Non-Human Primates

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2908-2908
Author(s):  
Samson Koelle ◽  
Chuanfeng Wu ◽  
Brian Li ◽  
Rong Lu ◽  
Robert E. Donahue ◽  
...  
Keyword(s):  
Stem Cell ◽  
B Cell ◽  
Cell Fate ◽  
High Throughput Sequencing ◽  
Conflicts Of Interest ◽  
Rhesus Macaques ◽  
Small Subset ◽  
Hematopoietic Stem ◽  
Over Time

Abstract By transplanting rhesus macaques with autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) labeled with lentivirally delivered high diversity oligonucleotide barcodes, we are able to track complex clonal contributions to hematopoiesis over time and across lineages via low cycle PCR and high-throughput sequencing of the inserted barcode. We reported on patterns of reconstitution for the first 9 months following engraftment in a recent publication (Wu et al, Cell Stem Cell, 2014). For the first 1 to 3 months short lived unilineage progenitors predominated, followed by increased clonal diversity and the emergence of first shared myeloid (My)/B clones, and then My/B/T clones. A major novel finding was a distinct ontogeny for NK cells, with the major CD16+ blood NK population showing non-overlapping origin with My/B and T lineages through 6-9 months. We now report on hematopoietic clonal patterns up to 23 months post-transplant in four animals. In all four animals, a group of high contributing NK clones contributed minimally to other lineages until 9 to 12 months, at which point these unilineage NK clones extinguished and a new generation of NK high contributors appeared. In two animals, these emerging clones continued to be either unilineage or highly biased towards the NK lineage, while in two other animals, they began to contribute highly to all lineages, a discrepancy which possibly derives from differences between animals in barcode marking level. Over the 17 and 23 months since transplant tracked in the two highly marked animals, 5.6±1.4 and 9±0.9 out of the top 10 NK contributing clones, respectively, were highly biased towards NK and away from other lineages, as determined by unsupervised hierarchical clustering of correlations between high contributing clones. Though shared My/B/T progenitors emerged after 6 months in both animals, 3.1±1.3 and 6.4±0.9 out of the T cell top 10 contributing clones were highly biased towards T cells and away from all other lineages up to 17 and 23 months. In contrast to the highly biased NK contributors, these T biased clones did not extinguish in the time surveyed so far, and more frequently were detected at low levels in the My/B lineages. The degree of bias of these high contributing clones towards the NK and T lineages was steady over time, suggesting that strong clonal bias is stable. The top 10 clones in NK contributed a significantly greater fraction of hematopoiesis (20±4% and 38±10%) than the top 10 clones in the T lineage (15±2% and 13±2%) in both animals (p=0.01, p<0.0001). In all lineages at all time points from 1 to 23 months, a small subset (14±3%) of clones detected at each individual time point contributed at least 50% of barcoded hematopoiesis. Polyclonality increased following initial hematopoietic reconstitution but started to fall around 6 months, reflecting the increased contribution of certain high contributing, totipotent clones. These clones did not extinguish after beginning to contribute, and have characteristics of long-term repopulating HSCs. We observed novel instability in the lineage bias of some individual HSPC contributions. For instance, an early totipotent clone which was a top 10 producer of granulocytes halted B cell contribution almost completely at 4.5 months, before regaining it by 5 months later and becoming the highest contributing clone in the entire animal. The high degree of bias towards and away from certain lineages observed in many long term repopulating HSCs is not necessarily in violation of hypothesized progenitor hierarchies, but it indicates the presence of poorly understood nuances in clonal control. In addition to the long-term dominant unbiased HSCs whose hematopoietic contribution increased over time, we noticed a cohort of clones biased towards a combined myeloid/B cell lineage. The varied clonal patterns observed between animals could result from individual differences in transplant progenitor composition or dose, or degree of recipient endogenous HSPC depletion. Understanding the lifespan and population dynamics of repopulating clones in macaques after transplantation is relevant for optimizing human hematopoietic stem cell transplants and also provides an approach for identifying progenitor populations, inferring mechanisms of cell fate control, and calculating rates of differentiation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Cell lineage and cell cycling analyses of the 4d micromere using live imaging in the marine annelid Platynereis dumerilii

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
B Duygu Özpolat ◽  
Mette Handberg-Thorsager ◽  
Michel Vervoort ◽  
Guillaume Balavoine
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Cell Fate ◽  
Cell Lineage ◽  
Growth Zone ◽  
Live Imaging ◽  
In Vivo Studies ◽  
Platynereis Dumerilii ◽  
Cell Cycling

Cell lineage, cell cycle, and cell fate are tightly associated in developmental processes, but in vivo studies at single-cell resolution showing the intricacies of these associations are rare due to technical limitations. In this study on the marine annelid Platynereis dumerilii, we investigated the lineage of the 4d micromere, using high-resolution long-term live imaging complemented with a live-cell cycle reporter. 4d is the origin of mesodermal lineages and the germline in many spiralians. We traced lineages at single-cell resolution within 4d and demonstrate that embryonic segmental mesoderm forms via teloblastic divisions, as in clitellate annelids. We also identified the precise cellular origins of the larval mesodermal posterior growth zone. We found that differentially-fated progeny of 4d (germline, segmental mesoderm, growth zone) display significantly different cell cycling. This work has evolutionary implications, sets up the foundation for functional studies in annelid stem cells, and presents newly established techniques for live imaging marine embryos.

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