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

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.

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Single cell transcriptional characterization of human megakaryocyte lineage commitment and maturation

10.1101/2020.02.20.957936 ◽

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.

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Elucidation of the EP defect in Diamond-Blackfan anemia by characterization and prospective isolation of human EPs

Blood ◽

10.1182/blood-2014-10-608042 ◽

2015 ◽

Vol 125 (16) ◽

pp. 2553-2557 ◽

Cited By ~ 17

Author(s):

Deena Iskander ◽

Bethan Psaila ◽

Gareth Gerrard ◽

Aristeidis Chaidos ◽

Hui En Foong ◽

...

Keyword(s):

Bone Marrow ◽

Human Bone ◽

Human Bone Marrow ◽

Diamond Blackfan Anemia ◽

Key Points ◽

Prospective Isolation

Key Points Identification and prospective isolation of EEP and LEP from human bone marrow (BM) facilitates the study of erythropoiesis. Quantitative and qualitative defects in EP underpinning erythropoietic failure in DBA are restored in steroid-responsive (SR) patients.

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Strategies to Identify Mesenchymal Stromal Cells in Minimally Manipulated Human Bone Marrow Aspirate Concentrate Lack Consensus

The American Journal of Sports Medicine ◽

10.1177/0363546521993788 ◽

2021 ◽

pp. 036354652199378

Author(s):

Severin Ruoss ◽

J. Todd Walker ◽

Chanond A. Nasamran ◽

Kathleen M. Fisch ◽

Conner J. Paez ◽

...

Keyword(s):

Bone Marrow ◽

Single Cell ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Bone Marrow Aspirate ◽

Transcriptional Profile ◽

Human Bone ◽

Human Bone Marrow ◽

Cell Transcriptome ◽

Single Cell Transcriptome

Background: There is a need to identify and quantify mesenchymal stromal cells (MSCs) in human bone marrow aspirate concentrate (BMAC) source tissues, but current methods to do so were established in cultured cell populations. Given that surface marker and gene expression change in cultured cells, it is doubtful that these strategies are valid to quantify MSCs in fresh BMAC. Purpose: To establish the presence, quantity, and heterogeneity of BMAC-derived MSCs in minimally manipulated BMAC using currently available strategies. Study Design: Descriptive laboratory study. Methods: Five published strategies to identify MSCs were compared for suitability and efficiency to quantify clinical-grade BMAC-MSCs and cultured MSCs at the single cell transcriptome level on BMAC samples being used clinically from 15 orthopaedic patients and on 1 cultured MSC sample. Strategies included (1) the guidelines by the International Society for Cellular Therapy (ISCT), (2) CD271 expression, (3) the Ghazanfari et al transcriptional profile, (4) the Jia et al transcriptional profile, and (5) the Silva et al transcriptional profile. Results: ISCT guidelines did not identify any MSCs in BMAC at the transcriptional level and only 1 in 9 million cells at the protein level. Of 12,850 BMAC cells, 9 expressed the CD271 gene. Only 116 of 396 Ghazanfari genes were detected in BMAC, whereas no cells expressed all of them. No cells expressed all Jia genes, but 25 cells expressed at least 13 of 22. No cells expressed all Silva genes, but 19 cells expressed at least 8 of 23. Most importantly, the liberalized strategies tended to identify different cells and most of them clustered with immune cells. Conclusion: Currently available methods need to be liberalized to identify any MSCs in fresh human BMAC and lack consensus at the single cell transcriptome and protein expression levels. These different cells should be isolated and challenged to establish phenotypic differences. Clinical Relevance: This study demonstrated that improved strategies to quantify MSC concentrations in BMAC for clinical applications are urgently needed. Until then, injected minimally manipulated MSC doses should be reported as rough estimates or as unknown.

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Single-cell RNA sequencing deconvolutes the in vivo heterogeneity of human bone marrow-derived mesenchymal stem cells

International Journal of Biological Sciences ◽

10.7150/ijbs.61950 ◽

2021 ◽

Vol 17 (15) ◽

pp. 4192-4206

Author(s):

Zun Wang ◽

Xiaohua Li ◽

Junxiao Yang ◽

Yun Gong ◽

Huixi Zhang ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Single Cell ◽

Rna Sequencing ◽

Human Bone ◽

Human Bone Marrow ◽

Single Cell Rna Sequencing

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Isolation of Nucleated Cells from Bone Marrow Aspirate v1

10.17504/protocols.io.bwrupd6w ◽

2021 ◽

Author(s):

Steven B. Wells ◽

Peter A. Szabo ◽

Nora Lam

Keyword(s):

Bone Marrow ◽

Single Cell ◽

Bone Marrow Aspirate ◽

Myeloid Cells ◽

Cell Suspensions ◽

Human Bone ◽

Human Bone Marrow ◽

Dilution Factor ◽

Density Centrifugation ◽

Defined Media

This protocol describes a method for the isolation of pan-lymphocytes, pan-myeloid cells, and progenitors from human bone marrow aspirate. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

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