scholarly journals Single cell transcriptional characterization of human megakaryocyte lineage commitment and maturation

2020 ◽  
Author(s):  
Fizzah A Choudry ◽  
Frederik Otzen Bagger ◽  
Iain C Macaulay ◽  
Samantha Farrow ◽  
Frances Burden ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Lineage Commitment ◽  
Hematopoietic Stem ◽  
Transcriptional Signature ◽  
Intermediate Progenitors ◽  
High Ploidy ◽  
Functional Relevance

AbstractIn the current understanding of adult bone marrow hematopoiesis, megakaryocytes (MKs) originate from cells immuno-phenotypically indistinguishable from hematopoietic stem cells (HSCs), bypassing intermediate progenitors. Here, we use single cell RNA sequencing to characterize HSCs and MKs from human bone marrow, to investigate MK lineage commitment and maturation. We identify two MK primed HSC clusters exhibiting unique differentiation kinetics, at least one of which is used in steady state and stress thrombopoiesis. By analyzing transcriptional signatures we show that human bone marrow MKs originate from MK primed HSC subpopulations, supporting the notion that these display exclusive priming for MK differentiation. We show that transcriptional programs change with increasing MK ploidy, where genes upregulated in high ploidy states may have functional relevance in platelet production. Finally, we highlight the presence of a specific transcriptional signature in MKs from individuals with myocardial infarction, supporting the aberration of MK differentiation in this thrombotic state.

Immunohistologic Identification and Quantification of Human Bone Marrow Hematopoietic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2301-2301
Author(s):  
Federico Quaini ◽  
Mirca Lazzaretti ◽  
Lorena Carranza ◽  
Francesca Ferraro ◽  
Sabrina Bonomini ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Simultaneous Detection ◽  
Hematopoietic Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Lineage Commitment ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Hematopoietic Stem ◽  
Bone Marrow Biopsies ◽  
Isolation And Characterization

Abstract A recent experimental and clinical observation on the use of bone marrow-derived stem/progenitor cells (BM-SC) to treat non-hematologic degenerative diseases has renovated the long-standing knowledge of their biology and function. However, the paradoxical lack of histological studies limits the identification of SC within the human BM, constraining their purification and further obscuring the sites where SC fate is decided. In an attempt to identify human hematopoietic BM-SC and their relationships with the tissue environment, bone marrow biopsies from 31 subjects without BM involvement by hemo-lymphoproliferative diseases were analysed with stringent morphometric criteria for the quantification of cells expressing CD117 (c-kit) and CD34, as candidate SC markers, and/or CD45, CD3, CD20, myeloperoxidase (MPOX), CD68, Glycophorin A (GlyA) and Factor VIII (vWF), as lineage commitment. Immunofluorescence combined with confocal examination was also performed to allow the simultaneous detection of multiple surface/cytoplasmic antigens together with transcription factors. Hematopoietic cells constituted 54% of the tissue among which MPOX, GlyA and vWF positive cells represented 43.8%, 13.5% and 0.06% of marrow cells, respectively. The frequency of CD34pos cells was 93.5±43.6 each 105 total nucleated cells; c-kit involved 2.7±2.6/105 cells and c-kit-CD34 co-expression was present in 1.14±0.98/105cells. When the incidence of CD45 was evaluated among these two cell populations only 15% of c-kitpos and 49% of CD34pos cells showed hematopoietic lineage commitment, indicating the presence in the human bone marrow of undifferentiated SC. The analysis of the expression of Terminal Deoxynucleotidyl Transferase (TdT) in hematopoietic cells demonstrated that 10% of c-kitpos, 20% of CD34pos and 0.62% of CD45pos cells possessed the nuclear transcription factor. The distribution of BM-SC was predominantly scattered throughout the tissue although interstitial and paratrabecular clusters were detected and are currently under intense scrutiny in search of BM-SC sites of activation. In a subset of patients, the histological analysis was complemented by the cytofluorimetric estimation of the same BM cells populations. The results of this study may contribute to the improvement in the isolation and characterization of BM-SC aimed to the identification of their niches and to an appropriate clinical application in the field of regenerative medicine.

A Stro-1+ human universal stromal feeder layer to expand/maintain human bone marrow hematopoietic stem/progenitor cells in a serum-free culture system

2006 ◽  
Vol 34 (10) ◽  
pp. 1353-1359 ◽  
Author(s):  
Raquel Gonçalves ◽  
Cláudia Lobato da Silva ◽  
Joaquim M.S. Cabral ◽  
Esmail D. Zanjani ◽  
Graça Almeida-Porada
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Culture System ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Feeder Layer ◽  
Hematopoietic Stem ◽  
Serum Free

scholarly journals The action of bryostatin on normal human hematopoietic progenitors is mediated by accessory cell release of growth factors

Blood ◽  
1990 ◽  
Vol 76 (4) ◽  
pp. 716-720 ◽  
Author(s):  
SJ Sharkis ◽  
RJ Jones ◽  
ML Bellis ◽  
GD Demetri ◽  
JD Griffin ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Single Cell ◽  
Cell Cultures ◽  
Bone Marrow Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Accessory Cell ◽  
Bryostatin 1 ◽  
Gm Csf

Abstract Since enrichment of human bone-marrow hematopoietic progenitors is becoming more feasible and since purified growth factors are now available, we sought to study the action of growth factors on CD34- positive enriched cultures of human bone-marrow cells. We tested the effect of recombinant human (rh) granulocyte-macrophage colony- stimulating factor (GM-CSF), rh interleukin-3 (IL-3), or a unique biologic response modifier, bryostatin 1, on the growth of purified CD34 cells obtained by limiting dilution in single-cell cultures. We have shown previously that bryostatin 1 stimulates both myeloid and erythroid progenitors of human origin in vitro. In this study both IL-3 and GM-CSF supported colony formation from 500, 100, or single-cell cultures at equivalent plating efficiences, suggesting a direct action of these factors on hematopoietic cell growth. Conversely, bryostatin 1 did not support the growth of CD34 cells in single-cell cultures, and the cloning efficiency increased with increasing the number of cells in the culture. To test whether the indirect action of bryostatin 1 might be mediated through the production of growth factors by accessory cells, studies were performed using antibodies directed against human IL-3 and GM-CSF in culture with bryostatin 1 and normal human bone- marrow cells. Results are consistent with the hypothesis that bryostatin 1 could have a stimulatory effect on the accessory cell populations to produce either IL-3 or GM-CSF. Further support for this notion was obtained by demonstrating that T cells, which are cells known to be able to produce IL-3 and GM-CSF, are stimulated by bryostatin 1 to express messenger RNA (mRNA) for specific growth factors, including GM-CSF. These results provide further support that bryostatin 1 may be a useful clinical agent to stimulate hematopoiesis in vivo.

scholarly journals Single-cell profiling of human bone marrow progenitors reveals mechanisms of failing erythropoiesis in Diamond-Blackfan anemia

2021 ◽  
Vol 13 (610) ◽  
Author(s):  
Deena Iskander ◽  
Guanlin Wang ◽  
Elisabeth F. Heuston ◽  
Chrysi Christodoulidou ◽  
Bethan Psaila ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Diamond Blackfan Anemia ◽  
Single Cell Profiling

scholarly journals Effects of in vitro purging with 4-hydroperoxycyclophosphamide on the hematopoietic and microenvironmental elements of human bone marrow

Blood ◽  
1985 ◽  
Vol 65 (3) ◽  
pp. 655-662
Author(s):  
S Siena ◽  
H Castro-Malaspina ◽  
SC Gulati ◽  
L Lu ◽  
MO Colvin ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Pluripotent Stem Cell ◽  
Cell Concentration ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Highly Sensitive

We describe the effects of 4-hydroperoxycyclophosphamide (4-HC) on the hematopoietic and stromal elements of human bone marrow. Marrow cells were exposed to 4-HC and then assayed for mixed (CFU-Mix), erythroid (BFU-E), granulomonocytic (CFU-GM), and marrow fibroblast (CFU-F) colony-forming cells and studied in the long-term marrow culture (LTMC) system. The inhibition of colony formation by 4-HC was dose and cell- concentration dependent. The cell most sensitive to 4-HC was CFU-Mix (ID50 31 mumol/L) followed by BFU-E (ID50 41 mumol/L), CFU-GM (ID50 89 mumol/L), and CFU-F (ID50 235 mumol/L). In LTMC, a dose-related inhibition of CFU-GM production was noted. Marrows treated with 300 mumol/L 4-HC were completely depleted of CFU-GM but were able to generate these progenitors in LTMC. Marrow stromal progenitors giving rise to stromal layers in LTMC, although less sensitive to 4-HC cytotoxicity, were damaged by 4-HC also in a dose-related manner. Marrows treated with 4-HC up to 300 mumol/L, gave rise to stromal layers composed of fibroblasts, endothelial cells, adipocytes, and macrophages. Cocultivation experiments with freshly isolated autologous hematopoietic cells showed that stromal layers derived from 4-HC- treated marrows were capable of sustaining the long-term production of CFU-GM as well as controls. In conclusion: (1) Hematopoietic progenitors cells, CFU-Mix, BFU-E, and CFU-GM, are highly sensitive to 4-HC, whereas marrow stromal progenitor cells are relatively resistant. (2) Marrows treated with 300 mumol/L 4-HC that are depleted of CFU-Mix, BFU-E, and CFU-GM can generate CFU-GM in LTMC, suggesting that most primitive hematopoietic stem cells (not represented by CFU-Mix) are spared by 4-HC up to this dose. (3) Consequently, the above colony assays are not suitable tools for predicting pluripotent stem cell survival after 4-HC treatment in vitro.

scholarly journals Expression of the YB5.B8 antigen (c-kit proto-oncogene product) in normal human bone marrow

Blood ◽  
1991 ◽  
Vol 78 (1) ◽  
pp. 30-37 ◽  
Author(s):  
LK Ashman ◽  
AC Cambareri ◽  
LB To ◽  
RJ Levinsky ◽  
CA Juttner
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Lymphoid Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Hematopoietic Stem ◽  
Normal Peripheral Blood ◽  
Normal Human ◽  
Normal Human Bone Marrow

Abstract The c-kit proto-oncogene product is a member of the family of growth factor receptors with intrinsic tyrosine kinase activity. In the mouse c-kit maps to the W locus, which is known to be of central importance in hematopoiesis. Monoclonal antibody (MoAb) YB5.B8, which was raised against peripheral blood blast cells from a patient with acute myeloid leukemia (AML), was recently shown to bind to the extracellular domain of the c-kit product. This antibody does not bind detectably to normal peripheral blood cells and identifies a sub-group of AML patients with poor prognosis. We have used MoAb YB5.B8 to study the expression of c- kit by normal human bone marrow cells by immunofluorescence and flow cytometry, and to isolate multipotential and erythroid colony-forming cells. In a series of 11 normal adult bone marrow specimens, MoAb YB5.B8 bound to 4.0% +/- 1.8% of the cells in the low-density fraction. Dual-labeling experiments were performed with YB5.B8, and CD33, CD34, and CD10 MoAbs. Three populations of cells binding YB5.B8 could be identified based on their pattern of coexpression of the other markers; ie, YB5.B8+/CD34+/CD33-, YB5.B8+/CD34+/CD33+ and YB5.B8+/CD34+/CD33+. These populations had distinctive two-dimensional light scatter characteristics and are likely to correspond to precursor colony- forming cells, colony-forming cells, and maturing mast cells, respectively. No cells binding both YB5.B8 and an MoAb to the early lymphoid marker CD10 were found, implying that most early lymphoid cells do not express c-kit. MoAbs to the c-kit protein should prove valuable in multimarker studies of human hematopoietic stem and progenitor cells. Definition of a reference range of c-kit expression in normal human bone marrow will provide a sound basis for further studies of this marker in diagnosis and prognosis in AML.

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