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Cell lineage tracking is a long-standing and unresolved problem in biology. Microfluidic technologies have the potential to address this problem, by virtue of their ability to manipulate and process single-cells in a rapid, controllable and efficient manner. Indeed, when coupled with traditional imaging approaches, microfluidic systems allow the experimentalist to follow single-cell divisions over time. Herein, we present a valve-based microfluidic system able to probe the decision-making processes of single-cells, by tracking their lineage over multiple generations. The system operates by trapping single-cells within growth chambers, allowing the trapped cells to grow and divide, isolating sister cells after a user-defined number of divisions and finally extracting them for downstream transcriptome analysis. The platform incorporates multiple cell manipulation operations, image processing-based automation for cell loading and growth monitoring, reagent addition and device washing. To demonstrate the efficacy of the microfluidic workflow, 6C2 (chicken erythroleukemia) and T2EC (primary chicken erythrocytic progenitors) cells are tracked inside the microfluidic device over two generations, with a cell viability rate in excess of 90%. Sister cells are successfully isolated after division and extracted within a 500 nL volume, which is compatible with downstream single-cell RNA sequencing analysis.

Additional file 1: Figure S1. [UMAP visualization of scRT-qPCR and scRNAseq data]. Figure S2. [PCA of scRT-qPCR and scRNAseq data]. Figure S3. [Half-life of mRNA table]. Figure S4. [Histograms of viability rate during reversion and differentiation processes]. Figure S5. [Summary table of scRNAseq data filtering steps and threshold values for each step].

Chronic myelogenous leukemia arises from the transformation of hematopoietic stem cells by the BCR-ABL oncogene. Though transformed cells are predominantly BCR-ABL-dependent and sensitive to tyrosine kinase inhibitor treatment, some BMPR1B+ leukemic stem cells are treatment-insensitive and rely, among others, on the bone morphogenetic protein (BMP) pathway for their survival via a BMP4 autocrine loop. Here, we further studied the involvement of BMP signaling in favoring residual leukemic stem cell persistence in the bone marrow of patients having achieved remission under treatment. We demonstrate by single-cell RNA-Seq analysis that a sub-fraction of surviving BMPR1B+ leukemic stem cells are co-enriched in BMP signaling, quiescence and stem cell signatures, without modulation of the canonical BMP target genes, but enrichment in actors of the Jak2/Stat3 signaling pathway. Indeed, based on a new model of persisting CD34+CD38- leukemic stem cells, we show that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways. Interestingly, we reveal that only the BMPR1B+ cells adhering to stromal cells display a quiescent status. Surprisingly, this quiescence is induced by treatment, while non-adherent BMPR1B+ cells treated with tyrosine kinase inhibitors continued to proliferate. The subsequent targeting of BMPR1B and Jak2 pathways decreased quiescent leukemic stem cells by promoting their cell cycle re-entry and differentiation. Moreover, while Jak2-inhibitors alone increased BMP4 production by mesenchymal cells, the addition of the newly described BMPR1B inhibitor (E6201) impaired BMP4-mediated production by stromal cells. Altogether, our data demonstrate that targeting both BMPR1B and Jak2/Stat3 efficiently impacts persisting and dormant leukemic stem cells hidden in their bone marrow microenvironment.

Additional file with several tables and figures related to in vitro genes parameters and waves estimation, in silico benchmarking and in vitro GRN candidates fit distance distribution. (PDF 312 kb)

Additional file 1: Table S1. Statistical analysis of gene expression according to the stained or the unstained condition. Statistical tests (Wilcoxon) were performed for each gene between their expression in stained and unstained condition. A Bonferroni correction was applied in p-values for multiple tests.

National audience; The v-erbA oncogene, carried by the Avian Erythroblastosis Virus, derives from the c-erbAalpha proto-oncogene which encodes the nuclear receptor for the thyroid hormone triiodothyronine (T3). In vitro, v-ErbA transforms erythroid progenitors by blocking their differentiation. It has been proposed that v-ErbA acts as a transcriptional repressor for genes normally activated by T3 and retinoic acid (RA), upregulated during the differentiation process. However, v-ErbA target genes responsible for transformation have yet to be identified. We used Serial Analysis of Gene Expression (SAGE) to analyze the transcriptome of avian erythroid progenitors (T2ECs), the natural target cells of v-erbA, expressing either an oncogenic form or a non-transforming form of verbA. The comparison of these two libraries revealed 83 genes differentially expressed between these two conditions. So far, the differential expression for 16 of them has been confirmed by real-time PCR on multiple independent repetitions. We observed that, among these v-ErbA target genes, some are activated by T3 and RA. This confirms that activation by T3 and RA receptors is indeed inhibited by v-ErbA. However, the expression of a vast majority of v-ErbA target genes did not vary in response to T3 and RA. These results suggest that v-ErbA must also act by T3- and RA-independent mechanisms in the transformation process. Furthermore, most v-erbA target genes do not vary during the differentiation process, in contrast to the expected role of v-erbA. In order to determine which major functions are deregulated by v-ErbA, we clustered the target genes identified according to the cellular function encoded by their corresponding proteins. We found that many of them are involved in the protein translation process. In order to understand the molecular mechanisms responsible for the coordinated variation of the v-ErbA target gene, we analyzed their promoter sequences and found the presence of c-myb binding sites as a signature motif of v-erbA target genes. This suggests a role for c-myb in the v-erbA-induced transformation process. Altogether, these studies demonstrate the involvement of new mechanisms pointing toward an unanticipated complexity of v-erbA oncogene action.