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Multiparameter analysis of timelapse imaging reveals kinetics of megakaryocytic erythroid progenitor clonal expansion and differentiation.

ABSTRACT: Single-cell assays have enriched our understanding of hematopoiesis and, more generally, stem and progenitor cell biology. However, these single-end-point approaches provide only a static snapshot of the state of a cell. To observe and measure dynamic changes that may instruct cell fate, we developed an approach for examining hematopoietic progenitor fate specification using long-term (> 7-day) single-cell time-lapse imaging for up to 13 generations with in situ fluorescence staining of primary human hematopoietic progenitors followed by algorithm-assisted lineage tracing. We analyzed progenitor cell dynamics, including the division rate, velocity, viability, and probability of lineage commitment at the single-cell level over time. We applied a Markov probabilistic model to predict progenitor division outcome over each generation in culture. We demonstrated the utility of this methodological pipeline by evaluating the effects of the cytokines thrombopoietin and erythropoietin on the dynamics of self-renewal and lineage specification in primary human bipotent megakaryocytic-erythroid progenitors (MEPs). Our data support the hypothesis that thrombopoietin and erythropoietin support the viability and self-renewal of MEPs, but do not affect fate specification. Thus, single-cell tracking of time-lapse imaged colony-forming unit assays provides a robust method for assessing the dynamics of progenitor self-renewal and lineage commitment.

SUBMITTER: Scanlon VM

PROVIDER: S-EPMC9519589 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Multiparameter analysis of timelapse imaging reveals kinetics of megakaryocytic erythroid progenitor clonal expansion and differentiation.

Scanlon Vanessa M VM Thompson Evrett N EN Lawton Betty R BR Kochugaeva Maria M Ta Kevinminh K Mayday Madeline Y MY Xavier-Ferrucio Juliana J Kang Elaine E Eskow Nicole M NM Lu Yi-Chien YC Kwon Nayoung N Laumas Anisha A Cenci Matthew M Lawrence Kalyani K Barden Katie K Larsuel Shannon T ST Reed Fiona E FE Peña-Carmona Gabriela G Ubbelohde Ashley A Lee June P JP Boobalan Shakthi S Oppong Yvette Y Anderson Rachel R Maynard Colby C Sahirul Kaylie K Lajeune Callista C Ivathraya Varsha V Addy Tiffany T Sanchez Patricia P Holbrook Colin C Van Ho Andrew Tri AT Duncan James S JS Blau Helen M HM Levchenko Andre A Krause Diane S DS

Scientific reports 20220928 1

Single-cell assays have enriched our understanding of hematopoiesis and, more generally, stem and progenitor cell biology. However, these single-end-point approaches provide only a static snapshot of the state of a cell. To observe and measure dynamic changes that may instruct cell fate, we developed an approach for examining hematopoietic progenitor fate specification using long-term (> 7-day) single-cell time-lapse imaging for up to 13 generations with in situ fluorescence staining of primary ...[more]

PMID: 36171423

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Project description:Programmed cell death-2 (PDCD2) protein is enriched in embryonic, hematopoietic, and neural stem cells, however, its function in stem/progenitor cell differentiation is unclear. We investigated the effects of PDCD2 knockdown on the development and differentiation of hematopoietic progenitor cells (HPC). CD34(+) cells derived from normal human bone marrow and K562 leukemic cells were effectively transduced with short-hairpin RNA to knockdown PDCD2. Colony-forming assays were used to investigate the effects of PDCD2 loss on HPC clonogenic potential and on 12-O-tetradecanoyl-phorbol-13-acetate-and arabinofuranosylcytosine-induced terminal differentiation. In CD34(+) clonogenic progenitors, PDCD2 knockdown decreased the total number of colony-forming units, increased the number of colony-forming units-granulocyte-erythroid-macrophage-megakaryocyte and burst-forming unit-erythroid primitive colonies, and decreased the number of burst-forming unit-erythroid mature colonies. Similar results were observed in K562 cells, suggesting that PDCD2 is important for HPC differentiation and/or survival, and for erythroid lineage commitment. Furthermore, 12-O-tetradecanoyl-phorbol-13-acetate-induced megakaryocytic differentiation and proliferation of K562 cells was not affected by PDCD2 knockdown. In contrast, arabinofuranosylcytosine-induced erythroid differentiation of K562 cells was significantly reduced with PDCD2 knockdown, with no effect on cell proliferation. The effects of PDCD2 knockdown were attributed to a cell cycle arrest at G(0)/G(1), along with increased messenger RNA expression of early progenitor factors c-MYB and GATA-2, and decreased expression of erythroid factors GATA-1, EpoR, and ?-globin. We conclude that PDCD2 loss of function(s) impedes erythroid differentiation by inducing cell cycle arrest and increasing expression of early hematopoietic progenitor factors. These findings suggest that PDCD2 has a novel regulatory role in human hematopoiesis and is essential for erythroid development.

Project description:BackgroundProper control of the lineage bias of megakaryocytic and erythroid progenitor cells (MEPs) is of significant importance, the disorder of which will lead to abnormalities in the number and function of platelets and erythrocytes. Unfortunately, the signaling pathways regulating MEP differentiation largely remain to be elucidated. This study aimed to analyze the role and the underlying molecular mechanism of miR-1915-3p in megakaryocytic and erythroid differentiation.MethodsWe utilized miRNA mimics and miRNA sponge to alter the expression of miR-1915-3p in megakaryocytic and/or erythroid potential cells; siRNA and overexpression plasmid to change the expression of SOCS4, a potential target of miR-1915-3p. The expression of relevant surface markers was detected by flow cytometry. We scanned for miR-1915-3p target genes by mRNA expression profiling and bioinformatic analysis, and confirmed the targeting by dual-luciferase reporter assay, western blot and gain- and lost-of-function studies. One-way ANOVA and t-test were used to analyze the statistical significance.ResultsIn this study, overexpression or knockdown of miR-1915-3p inhibited or promoted erythroid differentiation, respectively. Accordingly, we scanned for miR-1915-3p target genes and confirmed that SOCS4 is one of the direct targets of miR-1915-3p. An attentive examination of the endogenous expression of SOCS4 during megakaryocytic and erythroid differentiation suggested the involvement of SOCS4 in erythroid/megakaryocytic lineage determination. SOCS4 knockdown lessened erythroid surface markers expression, as well as improved megakaryocytic differentiation, similar to the effects of miR-1915-3p overexpression. While SOCS4 overexpression resulted in reversed effects. SOCS4 overexpression in miR-1915-3p upregulated cells rescued the effect of miR-1915-3p.ConclusionsmiR-1915-3p acts as a negative regulator of erythropoiesis, and positively in thrombopoiesis. SOCS4 is one of the key mediators of miR-1915-3p during the differentiation of MEPs.

Project description:NKL homeobox genes encode transcription factors that impact normal development and hematopoietic malignancies if deregulated. Recently, we established an NKL-code that describes the physiological expression pattern of eleven NKL homeobox genes in the course of hematopoiesis, allowing evaluation of aberrantly activated NKL genes in leukemia/lymphoma. Here, we identify ectopic expression of NKL homeobox gene NKX2-4 in an erythroblastic acute myeloid leukemia (AML) cell line OCI-M2 and describe investigation of its activating factors and target genes. Comparative expression profiling data of AML cell lines revealed in OCI-M2 an aberrantly activated program for endothelial development including master factor ETV2 and the additional endothelial signature genes HEY1, IRF6, and SOX7. Corresponding siRNA-mediated knockdown experiments showed their role in activating NKX2-4 expression. Furthermore, the ETV2 locus at 19p13 was genomically amplified, possibly underlying its aberrant expression. Target gene analyses of NKX2-4 revealed activated ETV2, HEY1, and SIX5 and suppressed FLI1. Comparative expression profiling analysis of public datasets for AML patients and primary megakaryocyte-erythroid progenitor cells showed conspicuous similarities to NKX2-4 activating factors and the target genes we identified, supporting the clinical relevance of our findings and developmental disturbance by NKX2-4. Finally, identification and target gene analysis of aberrantly expressed NKX2-3 in AML patients and a megakaryoblastic AML cell line ELF-153 showed activation of FLI1, contrasting with OCI-M2. FLI1 encodes a master factor for myelopoiesis, driving megakaryocytic differentiation and suppressing erythroid differentiation, thus representing a basic developmental target of these homeo-oncogenes. Taken together, we have identified aberrantly activated NKL homeobox genes NKX2-3 and NKX2-4 in AML, deregulating genes involved in megakaryocytic and erythroid differentiation processes, and thereby contributing to the formation of specific AML subtypes.

Dataset Information

Multiparameter analysis of timelapse imaging reveals kinetics of megakaryocytic erythroid progenitor clonal expansion and differentiation.

Publications

Multiparameter analysis of timelapse imaging reveals kinetics of megakaryocytic erythroid progenitor clonal expansion and differentiation.

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