Human Functional and Tunable in Vitro Model of Healthy and Multiple Myeloma Haematopoietic Niches

Introduction

Bone marrow (BM) niches regulate the fate and behavior of hematopoietic stem and progenitor cells (HSPCs) through cellular interactions and the secretion of soluble factors. The complexity of niche biology and its interaction with hematopoietic cells is well described in mice but it is only partially understood in humans. Here, we develop a novel, fully human hematopoietic niche model to improve our understanding of the human BM microenvironment and to study the human hematopoiesis and its commitment to myeloid and lymphoid lineages in health and multiple myeloma (MM) disease.

Methods

The niche models were generated using a single batch of either healthy or MM BM samples. Initially, pericyte-like stromal and endothelial cells were selected and amplified, then they were differentiated using a patented multi-differentiation medium (Adipocytic Osteoblastic Vascular medium/AOV medium) to mimic the different non-hematopoietic medullary compartments. The resulting medullary stroma was characterized through genetic (RT-qPCR) and protein approaches (immunofluorescence, ELISA assays) to evaluate cellular commitment towards the osteoblastic (RUNX2, OSX, BSP), adipocytic (PPARγ, LPL, ADIPOQ), and vascular (CD31) lineages, as well as the expression and secretion of pro-hematopoietic factors (CXCL12, SCF, LEPR, ANGPT1, VCAM1, OPN). After co-cultures were established by adding CD34+ cells sorted from cord blood (CB) or adult BM to fully differentiated stroma and cultured for 14 to 28 days without adding any exogenous cytokines. Co-cultures were treated with recombinant interleukin-1β (IL-1β) to simulate an inflammatory environment. The hematopoietic compartment was analyzed by flow cytometry to characterize and quantify the hematopoietic subpopulations.

Results

We generated a comprehensive medullary stroma featuring key non-hematopoietic cells, including mesenchymal stromal cells, osteo-progenitors, adipo-progenitors, endothelial cells from BM human healthy and MM samples. This mimetic human stroma, expressing crucial pro-hematopoietic factors such as CXCL-12, SCF and ANGPT1 successfully supported the proliferation of hematopoietic stem cell compartment (HSC and MPP) and drove hematopoietic differentiation. All lineage progenitors (MLP, GMP, MEP, CMP, CLP) and differentiated immune cells (granulocytes, monocytes and T lymphocytes) were produced in our hematopoietic niche model without additional cytokines. The addition of IL-1β, a key medullary inflammatory cytokine, skewed the differentiation in favor of the myeloid lineage, similar to in vivo response in mice. This skewing towards myeloid lineage was observed with both CB and BM sorted CD34+ cells in IL-1β treated co-culture, proving the key role of the microenvironment in the education and fate of HSPC. We used this model in the pathological context to better characterize the BM niches defects in MM. The generated stroma from MM BM samples exhibited reduced gene expression of osteoblastic, adipocytic, and vascular lineage markers, while pro-hematopoietic factor expression remained unchanged compared to the healthy condition. For the first time, co-culture experiments showed a perturbation of hematopoietic tree in MM, uncovering perturbations in the fine-tuning regulation of the hematopoietic process under pathological conditions.

Conclusion

This advanced, functional, and tunable hematopoietic niche model, derived from single-batch primary cells, offers a novel methodological approach for exploring and deciphering human hematopoiesis in healthy and disease contexts.