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Conditionally immortalised leukaemia initiating cells co-expressing Hoxa9/Meis1 demonstrate microenvironmental adaptation properties ex vivo while maintaining myelomonocytic memory.

ABSTRACT: Regulation of haematopoietic stem cell fate through conditional gene expression could improve understanding of healthy haematopoietic and leukaemia initiating cell (LIC) biology. We established conditionally immortalised myeloid progenitor cell lines co-expressing constitutive Hoxa9.EGFP and inducible Meis1.dTomato (H9M-ciMP) to study growth behaviour, immunophenotype and morphology under different cytokine/microenvironmental conditions ex vivo upon doxycycline (DOX) induction or removal. The vector design and drug-dependent selection approach identified new retroviral insertion (RVI) sites that potentially collaborate with Meis1/Hoxa9 and define H9M-ciMP fate. For most cell lines, myelomonocytic conditions supported reversible H9M-ciMP differentiation into neutrophils and macrophages with DOX-dependent modulation of Hoxa9/Meis1 and CD11b/Gr-1 expression. Here, up-regulation of Meis1/Hoxa9 promoted reconstitution of exponential expansion of immature H9M-ciMPs after DOX reapplication. Stem cell maintaining conditions supported selective H9M-ciMP exponential growth. H9M-ciMPs that had Ninj2 RVI and were cultured under myelomonocytic or stem cell maintaining conditions revealed the development of DOX-dependent acute myeloid leukaemia in a murine transplantation model. Transcriptional dysregulation of Ninj2 and distal genes surrounding RVI (Rad52, Kdm5a) was detected. All studied H9M-ciMPs demonstrated adaptation to T-lymphoid microenvironmental conditions while maintaining immature myelomonocytic features. Thus, the established system is relevant to leukaemia and stem cell biology.

SUBMITTER: Stahlhut M

PROVIDER: S-EPMC7935976 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Conditionally immortalised leukaemia initiating cells co-expressing Hoxa9/Meis1 demonstrate microenvironmental adaptation properties ex vivo while maintaining myelomonocytic memory.

Stahlhut Maike M Ha Teng Cheong TC Takmakova Ekaterina E Morgan Michael A MA Schwarzer Adrian A Schaudien Dirk D Eder Matthias M Schambach Axel A Kustikova Olga S OS

Scientific reports 20210305 1

Regulation of haematopoietic stem cell fate through conditional gene expression could improve understanding of healthy haematopoietic and leukaemia initiating cell (LIC) biology. We established conditionally immortalised myeloid progenitor cell lines co-expressing constitutive Hoxa9.EGFP and inducible Meis1.dTomato (H9M-ciMP) to study growth behaviour, immunophenotype and morphology under different cytokine/microenvironmental conditions ex vivo upon doxycycline (DOX) induction or removal. The ve ...[more]

PMID: 33674652

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Project description:BackgroundThoroughbred racehorse performance is largely influenced by a major quantitative trait locus at the myostatin (MSTN) gene which determines aptitude for certain race distances due to a promoter region insertion mutation influencing functional phenotypes in skeletal muscle. To develop an in vitro system for functional experiments we established three novel equine skeletal muscle cell lines reflecting the variation in phenotype associated with MSTN genotype (CC/II, CT/IN and TT/NN for SNP g.66493737C > T/SINE insertion 227 bp polymorphism). Primary equine skeletal muscle myoblasts, isolated from Thoroughbred horse gluteus medius, were conditionally immortalised and evaluated to determine whether cell phenotype and metabolic function were comparable to functional characteristics previously reported for ex vivo skeletal muscle isolated from Thoroughbred horses with each genotype.ResultsPrimary myoblasts conditionally immortalised with the temperature sensitive SV40TtsA58 lentivirus vector successfully proliferated and could revert to their primary cell phenotype and differentiate into multinucleated myotubes. Skeletal muscle fibre type, MSTN gene expression, mitochondrial abundance, and mitochondrial function of the three MSTN genotype cell lines, were consistent with equivalent characterisation of ex vivo skeletal muscle samples with these genotypes. Furthermore, addition of coenzyme Q10 (CoQ10) to the cell lines improved mitochondrial function, an observation consistent with ex vivo skeletal muscle samples with these genotypes following supplementation with CoQ10 in the diet.ConclusionsThe observation that the phenotypic characteristics and metabolic function of the cells lines are equivalent to ex vivo skeletal muscle indicates that this in vitro system will enable efficient and cost-effective analyses of equine skeletal muscle for a range of different applications including understanding metabolic function, testing of nutritional supplements, drug test development and gene doping test development. In the multi-billion-euro international Thoroughbred horse industry research advances in the biological function of skeletal muscle are likely to have considerable impact. Furthermore, this novel genotype-specific system may be adapted and applied to human biomedicine to improve understanding of the effects of myostatin in human physiology and medicine.

Project description:BackgroundChronic myelomonocytic leukaemia (CMML) is a clinically heterogeneous stem cell malignancy with overlapping features of myelodysplasia and myeloproliferation. Over 90% of patients carry mutations in epigenetic and/or splicing genes, typically detectable in the Lin-CD34+CD38- immunophenotypic stem cell compartment in which the leukaemia-initiating cells reside. Transcriptional dysregulation at the stem cell level is likely fundamental to disease onset and progression.MethodsWe performed single-cell RNA sequencing on 6826 Lin-CD34+CD38-stem cells from CMML patients and healthy controls using the droplet-based, ultra-high-throughput 10x platform.FindingsWe found substantial inter- and intra-patient heterogeneity, with CMML stem cells displaying distinctive transcriptional programs. Compared with normal controls, CMML stem cells exhibited transcriptomes characterized by increased expression of myeloid-lineage and cell cycle genes, and lower expression of genes selectively expressed by normal haematopoietic stem cells. Neutrophil-primed progenitor genes and a MYC transcription factor regulome were prominent in stem cells from CMML-1 patients, whereas CMML-2 stem cells exhibited strong expression of interferon-regulatory factor regulomes, including those associated with IRF1, IRF7 and IRF8. CMML-1 and CMML-2 stem cells (stages distinguished by proportion of downstream blasts and promonocytes) differed substantially in both transcriptome and pseudotime, indicating fundamentally different biology underpinning these disease states. Gene expression and pathway analyses highlighted potentially tractable therapeutic vulnerabilities for downstream investigation. Importantly, CMML patients harboured variably-sized subpopulations of transcriptionally normal stem cells, indicating a potential reservoir to restore functional haematopoiesis.InterpretationOur findings provide novel insights into the CMML stem cell compartment, revealing an unexpected degree of heterogeneity and demonstrating that CMML stem cell transcriptomes anticipate disease morphology, and therefore outcome.FundingProject funding was supported by Oglesby Charitable Trust, Cancer Research UK, Blood Cancer UK, and UK Medical Research Council.

Dataset Information

Conditionally immortalised leukaemia initiating cells co-expressing Hoxa9/Meis1 demonstrate microenvironmental adaptation properties ex vivo while maintaining myelomonocytic memory.

Publications

Conditionally immortalised leukaemia initiating cells co-expressing Hoxa9/Meis1 demonstrate microenvironmental adaptation properties ex vivo while maintaining myelomonocytic memory.

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