Size-Dependent Cortical Compaction Induces Metabolic Adaptation in Mesenchymal Stem Cell Aggregates

Abstract

This study reveals that multicellular aggregation induces metabolic reprogramming via mechanical compaction in lieu of formation of a hypoxic core. Utilizing biomechanical knowledge gained from planar culture, we set forth a novel three-dimensional (3D) model of size-dependent cortical compaction and demonstrated its role in metabolic reconfiguration. Ultimately, this study establishes mechanical compaction and its spatial gradients as key regulatory factors and design parameters in the development of 3D human adipose-derived mesenchymal stem cell aggregates.

Keywords: PI3K; adipose-derived stem cells; aggregation; aldolase A; metabolic reprogramming; oxygen tension.

FIG. 1.

Biomolecular gradient and diffusion in hASC. (A) Ru-dpp images of oxygen concentration in 10k, 30k, and 50k aggregates (scale bar is 200 μm). (B) Normalized radial oxygen profiles for size-dependent aggregates (represents average for size). (C) Oxygen consumption rates for planar, aggregate sizes. (D, E) Normalized radial and edge Thiele modulus for aggregates. Edge modulus model: (v_max = 1.0E-13 mol/cell/h, K_m = 2E-7 mol/m³, α = 21.54 mol/m³/h, β = −1.34E-13 mol/cell/h) Data represent the mean of at least three independent determinations; errors represent the standard error in the mean. *p < 0.05. hASCs, human adipose-derived stem cells.

FIG. 2.

Aggregation-induced reprogramming of metabolic profiles. (A–C) Plots of glucose consumed and lactate produce showing the level of metabolic reprogramming, along with the yield ratios. (D) Percentage of ATP as a result of aggregation. (E) Percentage of oxidative phosphorylation as determined by mitochondrial activity. (F) qPCR results for glycolytic genes. Data represent the mean of at least three independent determinations; errors represent the standard error in the mean. *p < 0.05. ATP, adenosine triphosphate; qPCR, quantitative polymerase chain reaction.

FIG. 3.

ASC aggregates show a size-dependent compaction. (A) (i) Immunohistochemical staining for nuclei and F-actin. (ii) Immunohistochemical staining for E-cadherin. (iii) Hematoxylin and eosin images for 10k, 30k, and 50k (scale bar is 100 μm). (iv) Cal AM 520 staining on whole ASC aggregates (scale bar is 200 μm) (B) Nuclear compaction as determined by observation of DAPI-stained nuclear aspect ratio. (C) Volume fraction of CalAM 520 stained aggregates as calculated by imaged radius. (D) Volume fraction of F-actin and E-cadherin as calculated by imaged radius. Data represent the mean of at least three independent determinations; errors represent the standard error in the mean. ASCs, adipose-derived mesenchymal stem cells.

FIG. 4.

Western blot analysis for mechanical signal transduction. (A) Blots and density reading for aggregation-induced and size-dependent mechanical phosphorylation with standardized tubulin. (B) qPCR results for cell–cell and cell–ECM proteins. Data represent the mean of at least three independent determinations; errors represent the standard error in the mean. ECM, extracellular matrix.

FIG. 5.

Images of ASC size-controlled mitochondrial fission and fusion. (A) Planar and dissociated aggregate cells were stained with Mitotracker to highlight the shape of the mitochondria (scale bars are 25 μm). (B) Mitochondrial elliptical aspect ratio was calculated by dividing the major axis by the minor axis. (C) Mitochondrial circularity was calculated by dividing the area by the perimeter squared. (D) qPCR results for mitochondrial fusion genes. (E) PCR results for mitochondrial fission genes. Data represent the mean of at least three independent determinations; errors represent the standard error in the mean. *p < 0.05. PCR, polymerase chain reaction.

FIG. 6.

AldoA hydrazine reduction assay results. (A) Size-dependent availability of AldoA. (B) Ratio of soluble to immobile AldoA for size-controlled aggregates. (C) qPCR for selected Aldolase response targets. (D) Western blot results for selected Aldolase and PI3K targets. Data represent the mean of at least three independent determinations; errors represent the standard error in the mean. AldoA, Aldolase A.