Assembling granular stem cell niches using microdroplet hydrogels

Replicating the cascade of signals necessary to control stem cell behavior remains a central challenge for the regenerative medicine community.

The hematopoietic system offers an ideal biological system to motivate the development of innovative technologies needed to accomplish this goal.

Hematopoiesis is the process where the body?s blood and immune cells are generated from a small number of hematopoietic stem cells (HSCs).

HSC quiescence, self-renewal, and differentiation take place in, and are regulated by, unique regions of the bone marrow termed niches.

Many innovations in stem cell engineering first focused on replicating constellations of extracellular matrix, biomolecular, or metabolic (e.g., hypoxia) signals within the niche.

For example, we developed microfluidic approaches to create gelatin hydrogels containing marrow-inspired gradients of stiffness, niche cells, and biomolecules for extended culture of 103-104 primary murine HSCs.

However, signaling between cohorts of different cell populations within the niche is also a critical regulator of stem cell expansion, quiescence, and lineage specification and may contribute to hematopoietic cancers.

We adapted our platform to show the kinetics of HSC-niche cell crosstalk can be manipulated via hydrogel network parameters to dramatically alter HSC fate.

We also developed bioinformatics tools to identify secretome signals generated by marrow mesenchymal stem cells (MSCs) that enhance retention of quiescent HSCs.

However, tools to study reciprocal signaling between multiple cell populations within an engineered stem cell niche remains limited by our ability to locally control the assembly, culture, and recovery of multicellular cohorts.

Conventional bulk hydrogels do not allow an avenue to tailor, or trace the evolution of, the local microenvironment surrounding unique cell subpopulations.

Our research community requires a new tissue engineering ecosystem that allows us to replicate dynamic, multicellular stem cell niches and also exploit recent advances in single-cell sequencing and bioinformatics.

The primary objective of this NIDDK Catalytic Tool and Technology Development project (R21 DK131751-01) is to develop underlying technology required to form a granular stem cell niche. Granular hydrogels are macroscale structures generated as jammed assemblies of microscale hydrogel particles.

To date they have been predominantly used as acellular hydrogel particles with cells cultured in the voids between particles.

Our innovative approach will encapsulate single marrow derived hematopoietic cells in distinct nanoliter-volume hydrogel microdroplets that can be rapidly formed, tailored for each discrete cell population, and non-toxically degraded.

We will use the short diffusion lengths of microdroplets to study of the convergence of matrix biophysical and metabolic signals on HSC fate. We address the high-risk, high-reward nature of this catalytic tool development project via the following aims: Aim 1. Establish a microdroplet artificial marrow unit cell.

We will generate essential features of a multi- cellular granular hydrogel niche.

We will formalize microdroplet fabrication parameters to encapsulate murine HSCs in nanoliter-volume hydrogels as distinct marrow unit cells.

We will benchmark patterns of in vitro HSC expansion in microdroplet niches in response to metabolic constraint (hypoxia).

We will diversify the microdroplet matrix via inclusion of marrow-mimetic hyaluronic acid, then use multi-parameter tools to quantify HA-induced shifts in HSC quiescence. Aim 2. Create granular assemblies of microdroplet hydrogels.

Ordered assembly, culture, then disassembly of hydrogel microdroplets provides the technical basis for a multicellular niche required to interrogate multicellular signaling.

We will form jammed assemblies of multiple families of acellular microdroplet hydrogels, evaluate their stability in culture, and demonstrate selective recovery of unique microdroplet hydrogel populations post culture. This revised aim has one subpart: Aim 2A. Manipulate the cohesion between particles to form and disassemble granular niches.

Impact. This proposed research is unified in its approach to develop innovative tools to mimic multicellular stem cell niches. HSCs in the bone marrow navigate diverse and dynamic matrix, metabolic, and cellular selection pressures.

We will develop critical tissue engineering infrastructure to study the integrated contribution of matrisome remodeling and multicellular signaling on HSC expansion and quiescence.

The well- characterized murine hematopoietic system provides a rigorous framework to evaluate and mimic ex vivo regulatory processes within niches whose rarity and complexity limit direct in vivo examination.

Consistent with score-driving criteria of the Catalytic Tool and Technology program, we will develop a novel, high-risk approach to generate multicellular stem cell niche analogs based on granular hydrogels.

We will control the assembly and disassembly of multicellular niches then employ analytical tools to study dynamic processes of matrix remodeling and HSC-niche cell crosstalk. Such studies are intractable in conventional bulk hydrogel cultures.

Efficient strategies to create ordered, hierarchical, and multicellular assemblies will be transformative to the NIDDK scientific and clinical community for studies of hematopoietic homeostasis, hematopoietic cancers, and for the development of new cancer therapies.