Towards Mimicking the Fetal Liver Niche: The Influence of Elasticity and Oxygen Tension on Hematopoietic Stem/Progenitor Cells Cultured in 3D Fibrin Hydrogels

Abstract

Hematopoietic stem/progenitor cells (HSPCs) are responsible for the generation of blood cells throughout life. It is believed that, in addition to soluble cytokines and niche cells, biophysical cues like elasticity and oxygen tension are responsible for the orchestration of stem cell fate. Although several studies have examined the effects of bone marrow (BM) niche elasticity on HSPC behavior, no study has yet investigated the effects of the elasticity of other niche sites like the fetal liver (FL), where HSPCs expand more extensively. In this study, we evaluated the effect of matrix stiffness values similar to those of the FL on BM-derived HSPC expansion. We first characterized the elastic modulus of murine FL tissue at embryonic day E14.5. Fibrin hydrogels with similar stiffness values as the FL (soft hydrogels) were compared with stiffer fibrin hydrogels (hard hydrogels) and with suspension culture. We evaluated the expansion of total nucleated cells (TNCs), Lin^-/cKit⁺ cells, HSPCs (Lin^-/Sca⁺/cKit⁺ (LSK) cells), and hematopoietic stem cells (HSCs: LSK- Signaling Lymphocyte Activated Molecule (LSK-SLAM) cells) when cultured in 5% O₂ (hypoxia) or in normoxia. After 10 days, there was a significant expansion of TNCs and LSK cells in all culture conditions at both levels of oxygen tension. LSK cells expanded more in suspension culture than in both fibrin hydrogels, whereas TNCs expanded more in suspension culture and in soft hydrogels than in hard hydrogels, particularly in normoxia. The number of LSK-SLAM cells was maintained in suspension culture and in the soft hydrogels but not in the hard hydrogels. Our results indicate that both suspension culture and fibrin hydrogels allow for the expansion of HSPCs and more differentiated progeny whereas stiff environments may compromise LSK-SLAM cell expansion. This suggests that further research using softer hydrogels with stiffness values closer to the FL niche is warranted.

Keywords: 3D cell encapsulation; elastic modulus; fetal liver niche; fibrin hydrogel; hematopoietic stem cells; oxygen tension.

Figure 1

Elastic moduli of fetal liver (FL) tissue and fibrin hydrogels: (a) representative force-indentation curves of murine E14.5 FL tissue and fibrin hydrogels and (b) calculated Young’s moduli of FL tissue and fibrin hydrogels. Boxplots show the distribution of Young’s modulus with boxes representing the interquartile ranges, while the mean is represented by the plus sign. Dots represent the average values from the different areas. The mean elastic modulus of FL tissue was calculated using 19 randomly selected areas from 3 independent samples. The mean elastic modulus of the 0.6 mg/mL fibrin hydrogels was calculated from 19 randomly selected areas from 4 independent samples. For 1.8 mg/mL fibrin hydrogels, 15 randomly selected areas from 3 independent samples were used. Statistical analysis was performed with nested t-tests: ** p < 0.01 and *** p < 0.001.

Figure 2

Quantification of total nucleated cells (TNCs) by NucleoCounter^® and hematopoietic stem/progenitor cell (HSPC) subgroups by flow cytometry retrieved from hydrogels (soft and hard) and suspension cultures (Susp) under hypoxia (H) and normoxia (N) relative to initial cell number over a culturing period of 10 days, shown as fold change of (a) TNCs, (b) Lin⁻/cKit⁺ cells, (c) Lin⁻/Sca⁺/cKit⁺ (LSK) cells, and (d) LSK-Signaling Lymphocyte Activated Molecule (SLAM) cells: data were obtained from 6 biological replicates in 3 independent experiments except for suspension cultures in hypoxia, which contained 4 biological replicates. Data are presented as mean ± S.E.M. Statistical analysis was performed with a three-way ANOVA followed by Tukey post hoc tests for multiple comparisons. Asterisks denote significance between the different culture conditions (soft hydrogels, hard hydrogels, and suspension culture) or between hypoxia and normoxia: * p < 0.05, ** p < 0.01, and *** p < 0.001. The plus signs denote significance between data collected at different time points against day 0: ⁺ p < 0.05, ⁺⁺ p < 0.01, and ⁺⁺⁺ p < 0.001.

Figure 3

Fibrin hydrogels support the viability of Lin⁻/cKit⁺ cells: (a) confocal images of live (green) and dead (red) cells in soft and hard fibrin hydrogels as well as in suspension culture (Susp) grown in hypoxia (H) or normoxia (N) and (b) quantification of viability of Lin⁻/cKit⁺ progeny using the NucleoCounter^®. Day 0 values are from the starting cell suspension. Scale bars are 200 μm, and white arrows indicate x- and y-axes for 2D images or x-, y-, and z-axes for 3D image stacks. Data were obtained from 2 biological replicates and are represented as mean ± S.E.M. Statistical analysis was performed with a three-way ANOVA followed by Tukey post hoc tests for multiple comparisons. The plus signs denote significance between data collected at different time points against day 0: ⁺⁺⁺ p < 0.001. Other significant differences between groups are indicated with ** p < 0.01 and *** p < 0.001.

Figure 4

Quantification of HSPC subgroups from LSK-enriched cell culture, retrieved from soft fibrin hydrogels and suspension culture (Susp) under hypoxic conditions (H) over a period of 14 days, shown as fold changes of (a) TNCs, (b) Lin⁻/cKit⁺ cells, (c) LSK cells, and (d) LSK-SLAM cells: Data were acquired from 1 biological sample (pool of 6 donor mice). Given the rapid proliferation observed, cells were harvested on day 7, and the expanded cells were re-cultured under the initial cell conditions for another 7 days.