Identification of Thymus Settling Progenitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2677-2677
Author(s):  
Benjamin A. Schwarz ◽  
Avinash Bhandoola
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Hematopoietic Stem Cells ◽  
Adult Life ◽  
Hematopoietic Stem ◽  
Adult Mice ◽  
Multipotent Progenitors ◽  
Pass Through ◽  
Adult Thymus

Abstract T cells develop in the thymus, but are ultimately derived from hematopoietic stem cells (HSCs) that reside in the bone marrow. In order to produce T cells throughout adult life, the thymus must be periodically seeded by bone marrow progenitors via the blood. The identity of progenitors that seed the adult thymus is unknown. To determine which bone marrow progenitors that have access to they thymus, we analyzed the blood of adult mice (Schwarz & Bhandoola, Nature Immunology 2004). We found that the only progenitors in blood with T lineage potential were lineage negative cells with high expression of Sca-1 and c-Kit (LSK). Such LSK cells in blood were potent T lineage progenitors, with the capacity to expand over a million fold in the thymus. Like the corresponding population in the bone marrow, the blood LSK population was heterogeneous, containing HSCs and downstream multipotent progenitors (MPPs) including RAG-expressing early lymphoid progenitors (ELPs) and CD62L+ cells. In order to determine which of these LSK subsets can settle in the thymus, we developed a quantitative assay for thymic seeding in normal adult mice. We find that the fraction of LSK cells that settle in the thymus from the blood is extremely small. Of the estimated 3,000 to 4,000 LSK cells that pass through the thymic circulation each day, less than 10 cells are able to settle in the thymus. Our data suggest that any decrease in thymic seeding, as may occur in aging, would lead to a decrease in total thymic output.

scholarly journals Hematopoietic Stem Cells Are Endowed with Erythroid Signature in Beta-Thalassemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-31
Author(s):  
Maria Rosa Lidonnici ◽  
Giulia Chianella ◽  
Francesca Tiboni ◽  
Matteo Barcella ◽  
Ivan Merelli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Gene Set Enrichment Analysis ◽  
Lineage Commitment ◽  
Erythroid Cell ◽  
Beta Thalassemia ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors

Background Beta-thalassemia (Bthal) is a genetic disorder due to mutations in the ß-globin gene, leading to a reduced or absent production of HbA, which interferes with erythroid cell maturation and limits normal red cell production. Patients are affected by severe anemia, hepatosplenomegaly, and skeletal abnormalities due to rapid expansion of the erythroid compartment in bone marrow (BM) caused by ineffective erythropoiesis. In a classical view of hematopoiesis, the blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. In human, novel purification strategies based on differential expression of CD49f and CD90 enrich for long-term (49f+) and short-term (49f−) repopulating hematopoietic stem cells (HSCs), with distinct cell cycle properties, but similar myeloid (My) and lymphoid (Ly) potential. In this view, it has been proposed that erythroid (Ery) and megakaryocytic (Mk) fates branch off directly from CD90-/49f− multipotent progenitors (MPPs). Recently, a new study suggested that separation between multipotent (Ery/My/Ly) long-term repopulating cells (Subset1, defined as CLEC9AhighCD34low) and cells with only My/Ly and no Ery potential (Subset2, defined as CLEC9AlowCD34high)occurs within the phenotypic HSC/MPP and CD49f+ HSCs compartment. Aims A general perturbed and stress condition is present in the thalassemic BM microenvironment. Since its impact on the hematopoietic cell subpopulations is mostly unknown, we will investigate which model of hematopoiesis/erythropoiesis occurs in Bthal. Moreover, since Beta-Thalassemia is an erythropoietic disorder, it could be considered as a disease model to study the 'erythroid branching' in the hematopoietic hierarchy. Methods We defined by immunophenotype and functional analysis the lineage commitment of most primitive HSC/MPP cells in patients affected by this pathology compared to healthy donors (HDs). Furthermore, in order to delineate the transcriptional networks governing hematopoiesis in Beta-thalassemia, RNAseq analysis was performed on sorted hematopoietic subpopulations from BM of Bthal patients and HDs. By droplet digital PCR on RNA purified from mesenchymal stromal cells of Bthal patients, we evaluated the expression levels of some niche factors involved in the regulation of hematopoiesis and erythropoiesis. Moreover, the protein levels in the BM plasma were analyzed by performing ELISA. Results Differences in the primitive compartment were observed with an increased proportion of multipotent progenitors in Bthal patients compared to HDs. The Subset1 compartment is actually endowed with an enhanced Ery potential. Focusing on progenitors (CD34+ CD38+) and using a new sorting scheme that efficiently resolved My, Ery, and Mk lineage fates, we quantified the new My (CD71-BAH1-/+) and Ery (CD71+ BAH1-/+) subsets and found a reduction of Ery subset in Bthal samples. We can hypothesize that the erythroid-enriched subsets are more prone to differentiate quickly due to the higher sensitivity to Epo stimuli or other bone marrow niche signals. Gene set enrichment analysis, perfomed on RNAseq data, showed that Bthal HSC/MPP presented negative enrichment of several pathways related to stemness and quiescence. Cellular processes involved in erythropoiesis were found altered in Bthal HSC. Moreover, some master erythroid transcription factors involved were overrepresented in Bthal across the hematopoietic cascade. We identified the niche factors which affect molecular pathways and the lineage commitment of Bthal HSCs. Summary/Conclusions Overall, these data indicate that Bthal HSCs are more cycling cells which egress from the quiescent state probably towards an erythroid differentiation, probably in response to a chronic BM stimulation. On the other hand,some evidences support our hypothesis of an 'erythroid branching' already present in the HSC pool, exacerbated by the pathophysiology of the disease. Disclosures No relevant conflicts of interest to declare.

ZFN-Driven Gene Editing Prevents HLA-A Expression On Hematopoietic Stem Cells -Improving The Chance Of Finding An HLA-Matched Donor

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1655-1655
Author(s):  
Hiroki Torikai ◽  
Tiejuan Mi ◽  
Sonny O Ang ◽  
Loren Gragert ◽  
Martin Maiers ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Hematopoietic Stem Cells ◽  
Donor Pool ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Immune Mediated ◽  
Matched Donor

Abstract Hematopoietic stem cells (HSCs) are administered (i) to restore hematopoiesis and immunity in the course of hematopoietic stem-cell transplantation (HSCT), (ii) as a replacement for inherited blood disorders and bone marrow failure, (iii) to regenerate cells of alternative lineages for restorative medicine, and (iv) as a source for generating specific hematopoietic cells (e.g., T cells, NK cells, and dendritic cells). However, the widespread application of allogeneic HSCs for humans is hampered by their immune-mediated destruction by host T cells recognizing mismatched HLA or by HLA-specific antibodies. Despite pre-banking umbilical cord blood (UCB) units and access to adult donors through the National Marrow Donor Program (NMDP), finding a suitable HLA-matched product is challenging for many recipients, especially those from ethnic minorities who are under-represented in the donor pool. The available donor pool would be markedly increased if donor HSCs were edited to eliminate expression of the HLA-A locus. Indeed, modeling from NMDP shows that the chance of an African American recipient finding a HLA-matched donor increases from 18% to 73% when matched for HLA-B, C and DR, instead of HLA-A, B, C and DR. We have previously shown that engineered zinc finger nucleases (ZFNs) can disrupt HLA-A expression in genetically edited T cells (Blood 2013). To extend this proof-of-concept to HSCs, we sought to disrupt HLA-A expression by introducing ZFNs targeting this locus. CD34+lineageneg HSCs (99% purity) were isolated using paramagnetic beads from UCB. Electro-transfer of in vitro transcribed mRNA encoding the HLA-A-specific ZFN generated 30% HLA-Aneg HSCs after one week ex vivo culture with defined cytokines (FLT3-L, SCF, TPO, and IL-6) and an aryl hydrocarbon receptor antagonist (stem reginin-1, SR-1). As expected, SR1 treatment maintained greater numbers of CD34+ cells (also CD34posCD38neg) in culture compared to controls. DNA sequence analysis revealed that HLA-Aneg HSCs encode the expected nucleotide changes at the ZFN target site. An in vivo engraftment assay, using NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, demonstrated that HLA-Aneg HSCs maintain the capability of engraftment and differentiation into HLA-Aneg hematopoietic cells (Figure). Thus, disruption of HLA-A expression in HSCs provides an appealing approach to increasing the chances for of finding HLA-matched donors and may broaden the clinical application of allogeneic HSCT. Furthermore, the ability to genetically edit HSCs has implications for (i) preventing immune-mediated recognition of HLA-disparate HSC and (ii) preventing immune mediated recognition of self-antigens. Engraftment of HLA-A2neg HSCs was evaluated in vivo. Data shown are flow-cytometry analysis of bone marrow obtained from NSG mice 16 weeks after HSC injection. HSC engraftment and HLA-A2 expression in NSG mice injected with un-modified HSCs (left panel) and HSCs treated with the HLA-A specific ZFNs (right panel) are shown. Data are gated on human CD45 positive cells. Figure Engraftment of HSCs modified by the HLA-A specific ZFNs in NSG mice. Figure. Engraftment of HSCs modified by the HLA-A specific ZFNs in NSG mice. Disclosures: Reik: Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment.

scholarly journals Human hematopoietic microenvironments

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250081
Author(s):  
Helene Bjoerg Kristensen ◽  
Thomas Levin Andersen ◽  
Andrea Patriarca ◽  
Klaus Kallenbach ◽  
Birgit MacDonald ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Treatment Modalities ◽  
Computer Assisted ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Murine Hematopoietic Stem Cell

Dormancy of hematopoietic stem cells and formation of progenitors are directed by signals that come from the bone marrow microenvironment. Considerable knowledge has been gained on the murine hematopoietic stem cell microenvironment, while less so on the murine progenitor microenvironment and even less so on these microenvironments in humans. Characterization of these microenvironments is decisive for understanding hematopoiesis and finding new treatment modalities against bone marrow malignancies in the clinic. However, it is equally challenging, because hematopoietic stem cells are difficult to detect in the complex bone marrow landscape. In the present study we are characterizing the human hematopoietic stem cell and progenitor microenvironment. We obtained three adjacent bone marrow sections from ten healthy volunteers. One was used to identify a population of CD34+/CD38- “hematopoietic stem cells and multipotent progenitors” and a population of CD34+/CD38+ “progenitors” based on immunofluorescence pattern/intensity and cellular morphology. The other two were immunostained respectively for CD34/CD56 and for CD34/SMA. Using the combined information we performed a non-computer-assisted quantification of nine bone marrow components (adipocytes, megakaryocytes, bone surfaces, four different vessel types (arteries, capillaries, sinusoids and collecting sinuses), other “hematopoietic stem cells and multipotent progenitors” and other “progenitors”) within 30 μm of “hematopoietic stem cells and multipotent progenitors”, “progenitors”, and “random cell profiles”. We show that the microenvironment of the “hematopoietic stem cells and multipotent progenitors” is significantly enriched in sinusoids and megakaryocytes, while the microenvironment of the “progenitors” is significantly enriched in capillaries, other “progenitors”, bone surfaces and arteries.

scholarly journals Hematopoietic stem cells self-renew symmetrically or gradually proceed to differentiation

2020 ◽  
Author(s):  
Melania Barile ◽  
Katrin Busch ◽  
Ann-Kathrin Fanti ◽  
Alessandro Greco ◽  
Xi Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Early Stage ◽  
Fate Mapping ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Lineage Differentiation ◽  
Adult Mice ◽  
Multipotent Progenitors ◽  
Cycle Dependent

SUMMARYIt is not known whether hematopoietic stem cells (HSCs) undergo symmetric or asymmetric cell divisions in the unperturbed bone marrow. Here, we integrate data from HSC fate mapping and cell-cycle-dependent labeling through mathematical inference and thus gain insight into how HSCs coordinate self-renewal with differentiation. We find that most HSC divisions in adult mice are symmetric self-renewing, replacing HSCs lost by direct differentiation and death, and slowly expanding the HSC population. This expansion maintains constant HSC output to multipotent progenitors (MPPs), despite declining HSC differentiation rate with age. We identify a linear hierarchy of differentiation states between tip HSCs and MPPs, where Tie2-driven HSC fate mapping fully covers the progression of the differentiating cells. A turning point from self-renewal to accelerated cell differentiation occurs between early-stage and late-stage MPPs, just before lineage differentiation becomes manifest in single-cell transcriptomes. This stem cell hierarchy precedes lineage differentiation and may limit mutation accumulation in the hematopoietic system.

scholarly journals Memory CD8 + T cells support the maintenance of hematopoietic stem cells in the bone marrow

Haematologica ◽  
2018 ◽  
Vol 103 (6) ◽  
pp. e230-e233 ◽  
Author(s):  
Sulima Geerman ◽  
Giso Brasser ◽  
Sudeep Bhushal ◽  
Fiamma Salerno ◽  
Natasja A. Kragten ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Hematopoietic Stem Cells ◽  
Cd8 T Cells ◽  
Hematopoietic Stem ◽  
Memory Cd8 T Cells

scholarly journals Reprogrammed Adult Human Endothelium into Hematopoietic Stem Cells Yields Functional T Cells In Vivo

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 169-169
Author(s):  
José Gabriel Barcia Durán ◽  
Tyler M. Lu ◽  
Raphael Lis ◽  
Shahin Rafii
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Hematopoietic Stem Cells ◽  
Constitutive Expression ◽  
Direct Conversion ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Human Endothelium

Abstract During development, the hematopoietic stem cells that go on to populate the bone marrow and give rise to all blood cell lineages emerge from a specialized endothelial subpopulation. We have previously harnessed this vestigial identity to achieve the direct conversion of adult endothelial cells (ECs) into long-term engraftable hematopoietic stem and progenitor cells (rEC-HSPCs); however, to date, we had only detected and characterized functional T cells that result from the transplantation, engraftment, and differentiation of mouse rEC-HSPCs (Lis, R. et al., Conversion of adult endothelium to immunocompetent haematopoietic stem cells. Nature, 545:439-445. 2017). We first reprogrammed adult human endothelium using lentiviral vectors encoding FOSB, GFI1, SPI1, and RUNX1 (FGRS) under constitutive promoters and an inductive vascular niche (Sandler V. et al., Reprogramming of human endothelium into hematopoietic cells requires vascular niche induction. Nature, 511:312-8. 2014). The resulting rEC-HSPCs maintained exogenous expression of the four transcription factors for over 20 weeks post-transplantation into immuno-compromised NSG mice. Constitutive expression of Spi1, however, has been shown to hinder lymphoid differentiation in vivo by blocking T lymphopoiesis (Anderson, M. et al. Constitutive expression of PU.1 in fetal hematopoietic progenitors blocks T cell development at the pro-T cell stage. Immunity 16:285-296. 2002). In addition, mice of the NSG strain cannot educate native B or T cells to maturity. Our system of constitutive exogenous FGRS expression was therefore unable to confer transplanted immuno-compromised mice the ability to generate an adaptive immune response. Here, we obtained human rEC-HSPCs making use of (i) doxycycline-inducible vectors to temporarily overexpress FGRS and (ii) transgenic substrains of NSG mice (one carrying human stem cell factor, granulocyte/macrophage colony-stimulating factor, and interleukin 3; the other, human major histocompatibility complex class I as well as beta-2 microglobulin) for transplantation assays. We show that human rEC-HSPCs engraft primary- and secondary-transplanted mice for over a year at levels of up to 20% in the spleen or bone marrow. Engrafted cells differentiate into all blood lineages including phenotypically and functionally mature T cells in the absence of exogenous FGRS expression in vivo. Notably, the resulting T cells undergo TCR rearrangement and are able to clear viral particles one week post-LCMV infection. Functional and phenotypic analyses are presented in juxtaposition with experiments using cord blood-transplanted mice. These results demonstrate that our present direct conversion strategy generates bona fide human hematopoietic stem cells from adult endothelial cells. Disclosures Rafii: Angiocrine Bioscience: Equity Ownership.

Increasing C/Ebpα Expression Regulates the Transition of Hematopoietic Stem Cells During Development.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1506-1506
Author(s):  
Min Ye ◽  
Hong Zhang ◽  
Pu Zhang ◽  
Daniel G. Tenen
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Induced Pluripotent Stem Cell ◽  
Gene Profiling ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Adult Mice ◽  
Induced Pluripotent

Abstract Abstract 1506 Poster Board I-529 During ontogeny, hematopoietic stem cells (HSCs) undergo a change from rapid dividing cells with high reconstitution ability to mainly quiescent cells with lower repopulation capacity. However, little is known about how this switch is regulated. Here we report that levels of C/EBPα, a transcription factor that is frequently disrupted in human acute myeloid leukemia, regulate the proliferation and self-renewal transition of HSCs during development. Loss of C/EBPα in adult mice resulted in a profound expansion of phenotypic HSCs and elevated proliferation rates. Limiting dilution transplantation to measure the frequency of competitive repopulation units (CRU) revealed an increase in the number of functional HSC in C/EBPα-/- mice. Serial transplantation of C/EBPα-/- bone marrow showed advanced reconstitution ability, indicating enhanced self-renewal ability. Interestingly, levels of C/EBPα in HSCs were significantly up-regulated 3 weeks after birth during which HSCs change from an actively cycling state to quiescence in bone marrow. When we conditionally inactivated C/EBPα in mice of different age, we observed a tight correlation between the age-specific levels of C/EBPα expression and the expansion of HSCs. Gene profiling analysis of C/EBPα-/- adult HSCs showed the up-regulation of oncogenes c-myc and n-myc, whose expression can regulate pluripotency and self-renewal of stem cells, as shown by recent induced pluripotent stem cell studies. Knocking down n-myc and c-myc expression in C/EBPα-/- HSCs using shRNA, we observed reduced proliferation and decreased colony formation in serial replating assay, which assesses the preservation of “self-renewal” in the progenitor cell compartment. Consistently, we observed down-regulation of n-myc in HSCs during the transition time, which is reciprocal to C/EBPα expression. Together, our data indicate C/EBPα as a key regulator of HSC self-renewal and proliferation during development, whose levels of expression might control the fetal to adult switch of HSC properties through regulating myc genes. Disclosures No relevant conflicts of interest to declare.

scholarly journals Engineering Precision Medicine to Increase Graft-Versus-Lymphoma Activity: Hematopoietic Stem Cells Modified with Chimeric Antigen Receptors

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2206-2206
Author(s):  
Satiro N. De Oliveira ◽  
Laurel C Truscott ◽  
Roy L Kao ◽  
Tzu-Ting Chiou
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Hematopoietic Stem Cells ◽  
Suicide Gene ◽  
Antigen Receptors ◽  
Chimeric Antigen Receptors ◽  
Hematopoietic Stem

Abstract Background: Patients with refractory or recurrent B-lineage hematological malignancies have less than 50% of chance of cure. Trials using autologous T-cells engineered with anti-CD19 chimeric antigen receptors (CAR) have demonstrated complete remissions even in chemotherapy-resistant malignancies, but the persistence of the cells is transient, limiting efficacy. Our hypothesis is modification of hematopoietic stem cells (HSC) with CAR will lead to persistent production of target-specific immune cells in multiple lineages, enhancing graft-versus-tumor activity and development of immunological memory. Design/Methods: Using CD19 as target, we generated second-generation CD28- and 4-1BB-costimulated CAR constructs for modification of human HSC for assessment in vitro and in vivo, using third-generation lentiviral vectors. Additionally, co-delivery of suicide gene systems was tested to allow ablation of gene-modified cells. Results: Gene modification of HSC with anti-CD19 CAR using lentiviral vectors did not impair differentiation or proliferation, and led to functional CAR-expressing cell progeny, at 40-50% transduction efficiency and engineered antigen-dependent cytotoxicity in myeloid, NK and T-cells. In vivo studies using humanized NSG engrafted with CAR-modified HSC demonstrated similar levels of humanization to non-modified HSC, with multilineage CAR-expressing cells present in bone marrow, spleen, blood and thymus in stable levels up to 44 weeks of life. No animals engrafted with CAR-modified HSC presented autoimmunity or inflammation. Ex vivo cells presented antigen-dependent cytotoxicity against targets. Mice engrafted with CAR-modified HSC had decreased CD19+ populations and successfully presented tumor growth inhibition and survival advantage at tumor challenge (55-60%). CAR-modified HSC led to development of T-cell effector memory and T-cell central memory phenotypes, confirming the development of long-lasting phenotypes due to directed antigen specificity. Mice humanized with gene-modified HSC presented significant ablation of gene-modified cells after treatment (p=0.002). Remaining gene-modified cells were close to background on flow cytometry and within two logs of decrease of vector copy numbers by ddPCR in mouse tissues. Conclusions: CAR modification of HSC for cancer immunotherapy is feasible. This approach can be applied to different cancers by adjusting target specificity and could be employed in the context of HSC transplantation to augment the anti-lymphoma activity. It also bears the possibility of decreased morbidity and mortality and offers alternative treatment for patients with no available sources for bone marrow transplantation, benefiting ethnic minorities. These results also give proof of principle for CAR-modified HSC regulated by suicide gene; further studies are needed to enable clinical translation. Disclosures No relevant conflicts of interest to declare.

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