Recreating the Hematopoietic Microenvironment in Vitro Using Microfluidic “on-a-Chip” Technology Enables the Study of Interactions of Normal and Malignant HSPC with Specific Bone Marrow Niches

Under normal conditions, hematopoietic stem/progenitor cells (HSPC) reside within specific bone marrow (BM) niches. These are comprised of an array of different cell types located strategically to provide a myriad of chemical signals and physical interactions that maintain HSPC and orchestrate the process of hematopoiesis. In human myeloid malignancies, the BM niche is remodeled by malignant cells, which displace resident HSPC, and create self-reinforcing malignant niches that drive disease progression, drug-resistance, and relapse. Attaining a mechanistic understanding of the major determinants of HSPC function in the human BM has been difficult, due to challenges associated with visualizing and modeling the complex niche environment in humans. The creation of accurate models of normal and malignant human BM niches would fill a critical gap, and enable the development of therapies targeting deviant cells and/or signaling pathways, to disrupt self-reinforcing malignant niches and reestablish normal hematopoiesis. We showed that microfluidic "on-a-chip" technologies using bioengineered 3D human tissue constructs, in the presence of physiological flow, can model normal physiology and disease processes and enable drug screening. Herein, we sought to exploit the high physiological relevance and single cell resolution offered by "on-a-chip" technology to recreate the BM microenvironment in vitro and study, in real-time, the interactions of normal and malignant HSPC with each specific niche cell type. To accomplish this objective, we constructed a microfluidic platform using polydimethylsiloxane (PDMS) and photolithography, in which microchannels allow continuous flow of media and HSPC through each of the specific niches, reproducing, in effect, a primitive circulatory system. To recreate the various BM niches, mesenchymal cells (Stro-1+; MSC), arterial endothelium (CD146+NG2+; AEC), and sinusoidal endothelium (CD146+NG2-; SEC) were magnetically-sorted from adult human BM. An aliquot of Stro-1+ cells was also induced to undergo osteogenic differentiation, to generate differentiated osteoblasts (OB). To create the in situ patterned 3D niche-on-a-chip (NOC), the chambers of the microfluidic device were filled with AEC, SEC, MSC, or OB embedded in a hyaluronic acid (HA)-based hydrogel precursor, and the cellularized constructs were formed by UV photopolymerization through the apertures of a photomask. Following flushing of uncrosslinked hydrogel precursor solution, the niche constructs were allowed to equilibrate for 3 days, under constant media flow using a four-channel precision microperistaltic pump. U937, MOLM13, and normal CD34⁺ cells were each labeled with Qtracker 605, and independently perfused into the main channel of a separate NOC, from which theyenter and evenly disperse into four equidistant chambers, each of which housed a distinct BM niche (AEC, SEC, MSC, or OB). LIVE/DEAD staining confirmed viability of the HSPC and niche cells for > 5 days following HSPC infusion, and immunocytochemistry demonstrated continued expression of appropriate phenotypic markers by each specific niche cell. At 24h post-infusion, U937 cells already exhibited a marked predilection for AEC, and this persisted throughout the 5-day observation period, with roughly 4.5-times more U937 cells engrafting within the AEC niche than the SEC or MSC niches, and 3-times more engrafting within the AEC niche than the OB niche. In contrast, MOLM13 cells exhibited a marked preference for the MSC and SEC niches, a moderate affinity for the OB niche, and engrafted only minimally within the AEC niche. In contrast to malignant cells, normal CD34+ HSPC engrafted preferentially within the SEC and OB niches, exhibited moderate engraftment within the MSC niche, and did not engraft the AEC niche. In conclusion, our studies establish the feasibility of using microfluidic "on-a-chip" technology to recreate the various niches within the BM microenvironment, and provide proof that this novel system can be used to study the interactions of normal and malignant HSPC with distinct cells of the niche. We are currently using this system to delineate the signaling pathways responsible for the observed preferential HSPC/niche cell interactions, with the ultimate goal of using this knowledge to develop more effective treatments for hematological malignancies and enhance engraftment following HSPC transplant.