Regulating dynamic signaling between hematopoietic stem cells and niche cells via a hydrogel matrix

Abstract

Hematopoietic stem cells (HSC) reside in unique bone marrow niches and are influenced by signals from surrounding cells, the extracellular matrix (ECM), ECM-bound or diffusible biomolecules. Here we describe the use of a three-dimensional hydrogel to alter the balance of HSC-generated autocrine feedback and paracrine signals generated by co-cultured niche-associated cells. We report shifts in HSC proliferation rate and fate specification in the presence of lineage positive (Lin⁺) niche cells. Hydrogels promoting autocrine feedback enhanced expansion of early hematopoietic progenitors while paracrine signals from Lin⁺ cells increased myeloid differentiation. We report thresholds where autocrine vs. paracrine cues alter HSC fate transitions, and were able to selectively abrogate the effects of matrix diffusivity and niche cell co-culture via the use of inhibitory cocktails of autocrine or paracrine signals. Together, these results suggest diffusive biotransport in three-dimensional biomaterials are a critical design element for the development of a synthetic stem cell niche.

Keywords: Autocrine; Hematopoietic; Hydrogel diffusivity; Paracrine; Signaling.

Fig. 1

Hematopoietic stem cell – Lin⁺ niche cell interactions within a hydrogel niche. (A) A schematic depicting biomolecular interactions between HSCs and niche cells encapsulated within a hydrogel matrix. P: paracrine signals generated by the niche cell population. A1: Fraction of HSC-generated autocrine signals that contribute to feedback loop. A2: Fraction of HSC-generated autocrine signals that diffuse away. (B) Schematic of expected effects of changes in niche cell density versus matrix diffusivity on the magnitude of Paracrine vs. Autocrine feedback reaching the HSC population. Magnitude of signal depicted by the arrow width and font size. (C) Representative image of the HSC differentiation hierarchy depicting the starting Lin⁻Sca1⁺cKit⁺ (LSK) fraction along with the early vs. late hematopoietic progenitor cell populations. LT-HSC: Long-term repopulating HSC (LSK·CD34⁻·Flk2⁻). ST-HSC: Short-term repopulating HSC. MPP: Multipotent progenitor (LSK·CD34⁻·Flk2⁻). CMP: common myeloid progenitor. MEP: megakaryocyte-erythrocyte progenitor. GMP: granulocyte-macrophage progenitor.

Fig. 2

Co-culture of LSK cells with Lin⁺ niche cells in liquid (2D) culture. (A) Viability of Lin⁻Sca1⁺cKit⁺ (LSK) HSC as a function of niche cell density. (B) Total number of LSK cells remaining as a function of niche cell density. (C)–(H) Fraction of the early and late hematopoietic progenitors as a function of LSK:niche cell co-culture density. LT-HSC: Long-term repopulating HSC (LSK·CD34⁻·Flk2⁻). ST-HSC: Short-term repopulating HSC. MPP: Multipotent progenitor (LSK·CD34⁻·Flk2⁻). CMP: common myeloid progenitor. MEP: megakaryocyte-erythrocyte progenitor. GMP: granulocyte-macrophage progenitor. *: p < 0.05. **: p < 0.01. ***: p < 0.005. NS: No significance.

Fig. 3

Comparative analysis of hematopoietic progenitor populations via flow cytometry as a function of hydrogel matrix conditions and niche cell co-culture density. (A) Early hematopoietic progenitor populations (LT-HSC, ST-HAS, MPP) increase with increasing niche cell density. (B) Representative schematic of the HSC differentiation hierarchy and heat map legend depicting changes in HSC:niche cell density (‘paracrine signal’) as well as hydrogel density (‘diffusivity’). (C) Heat map depicting relative changes in progenitor cell populations in 1 and 3 mg/mL collagen hydrogel conditions normalized against observed changes in 2D liquid (diffusion-unlimited) culture. Separate heat map scales are used for Early vs. Late hematopoietic progenitors to better demonstrate effects of niche cell and hydrogel density. *: p < 0.05 w.r.t. 2D culture. **: p < 0.01 w.r.t. 2D culture. ***: p < 0.001 w.r.t. 2D culture. #: p < 0.05 w.r.t. 1 mg/mL culture.

Fig. 4

Effect of the addition of paracrine-inhibitory cocktail on HSC response to Lin⁺ niche cells within the hydrogel environment. (A) A schematic depicting the effect of paracrine inhibition on HSCs in 1 mg/mL collagen co-cultures. HSC response was quantified via surface antigen expression using a modified heat map depiction of the effects of paracrine inhibition (right column) vs. culture in 1 mg/ml collagen (left column). Functional changes in HSC response were also profiled via CFU-assays (CFU-GEMM, CFU-GM, along with more mature Mk, E, G, M colonies). (B) Heat map-based analysis of effect of addition of paracrine inhibitor cocktails to HSC:Lin⁺ niche cell co-culture in paracrine-dominated (1 mg/ mL) hydrogel environments showing fold change in cell fractions both without (left column) and with (right column) paracrine inhibitors, with values normalized against 2D co-culture. (C) CFU assay showing effect of paracrine inhibition on progressively mature colonies, GEMM (most primitive) to E (most mature). *: p < 0.05 w.r.t. 2D culture. **: p < 0.01 w.r.t. 2D culture. ***: p < 0.001 w.r.t. 2D culture. NS: Not significant.

Fig. 5

Effect of the addition of autocrine-inhibitors on HSC response within the hydrogel environment. (A) A schematic hypothesizing the effects of autocrine inhibition in 3 mg/mL collagen co-cultures. HSC response was quantified via surface antigen expression using a modified heat map depiction of the effects of autocrine inhibition (right column) vs. culture in 3 mg/ml collagen (left column). Functional changes in HSC response were also profiled via CFU-assays (CFU-GEMM, CFU-GM, along with more mature Mk, E, G, M colonies). (B) Heat map-based analysis of effect of addition of autocrine inhibitor cocktails to HSC:Lin⁺ niche cell co-culture in autocrine-dominated (3 mg/mL) hydrogel environments showing fold change in cell fractions both without (left column) and with (right column) autocrine inhibitors, with values normalized against 2D co-culture. (C) CFU assay showing effect of paracrine inhibition on progressively mature colonies, GEMM (most primitive) to E (most mature). *: p < 0.05 w.r.t. 2D culture. **: p < 0.01 w.r.t. 2D culture. ***: p < 0.001 w.r.t. 2D culture. NS: Not significant.