A Human Bone Marrow 3D Model to Investigate the Dynamics and Interactions Between Resident Cells in Physiological or Tumoral Contexts.

The medullary niche is a complex ecosystem that is essential to maintain homeostasis for resident cells. Indeed, the bone marrow, which includes a complex extracellular matrix and various cell types, such as mesenchymal stem cells, osteoblasts, and endothelial cells, is deeply involved in hematopoietic stem cell regulation through direct cell-cell interactions, as well as cytokine production. To closely mimic this in vivo structure and conduct experiments reflecting the responses of the human bone marrow, several 3D models have been created based on biomaterials, relying primarily on primary stromal cells. Here, a protocol is described to obtain a minimal and standardized system that is easy to set up and provides features of bone marrow-like structure, which combines different cell populations including endothelial cells, and reflects the heterogeneity of in vivo bone marrow tissue. This 3D bone marrow-like structure-assembled using calcium phosphate-based particles and human cell lines, representative of the bone marrow microenvironment-allows the monitoring of a wide variety of biological processes by combining or replacing different primary cell populations within the system. The final 3D structures can then either be harvested for image analysis after fixation, paraffin-embedding, and histological/immunohistochemical staining for cell localization within the system, or dissociated to collect each cellular component for molecular or functional characterization.

The quiescent fraction of chronic myeloid leukemic stem cells depends on BMPR1B, Stat3 and BMP4-niche signals to persist in patients in remission.

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.