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Studies of mechanical signalling are typically performed by comparing cells cultured on soft and stiff hydrogel-based substrates. However, it is challenging to independently and robustly control both substrate stiffness and extracellular matrix tethering to substrates, making matrix tethering a potentially confounding variable in mechanical signalling investigations. Moreover, unstable matrix tethering can lead to poor cell attachment and weak engagement of cell adhesions. To address this, we developed StemBond hydrogels, a hydrogel in which matrix tethering is robust and can be varied independently of stiffness. We validate StemBond hydrogels by showing that they provide an optimal system for culturing mouse and human pluripotent stem cells. We further show how soft StemBond hydrogels modulate stem cell function, partly through stiffness-sensitive ERK signalling. Our findings underline how substrate mechanics impact mechanosensitive signalling pathways regulating self-renewal and differentiation, indicating that optimising the complete mechanical microenvironment will offer greater control over stem cell fate specification.

In many leukemia patients, a poor prognosis is attributed either to the development of chemotherapy resistance by leukemic stem cells (LSCs) or to the inefficient engraftment of transplanted hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM). Here, we build a 3D in vitro model system of bone marrow organoids (BMOs) that recapitulate several structural and cellular components of native BM. These organoids are formed in a high-throughput manner from the aggregation of endothelial and mesenchymal cells within hydrogel microwells. Accordingly, the mesenchymal compartment shows partial maintenance of its self-renewal and multilineage potential, while endothelial cells self-organize into an interconnected vessel-like network. Intriguingly, such an endothelial compartment enhances the recruitment of HSPCs in a chemokine ligand/receptor-dependent manner, reminiscent of HSPC homing behavior in vivo. Additionally, we also model LSC migration and nesting in BMOs, thus highlighting the potential of this system as a well accessible and scalable preclinical model for candidate drug screening and patient-specific assays.

Hydrogeological properties of fluid reservoirs in the brittle continental crust at 5 km have been deduced from hydraulic and chemical data provided by the Deep Heat Mining well Basel-1 in the south of the Upper Rhine rift valley (central Europe, Switzerland). The investigation was challenging because no direct temperature logs or fluid samples from the undisturbed reservoir exist. However, the properties of the undisturbed reservoir have been reliably reconstructed from short time hydraulic tests and the evolution of outflow water composition. The rock of the open hole sections (4629–5000 m) is predominantly coarse-grained undeformed poorly fractured quartz-monzodiorite. The permeability k = 5.8 × 10–18 m2 is characteristic for plutonic basement at 5 km depth. Fluid flow is restricted to few steeply dipping fracture zones in this section. Outflow water triggered by massive injection of river water contains predominantly NaCl. The total of dissolved solids (TDS) in the pristine reservoir at depth is about 45 g kg−1. The origin of the high salinity is probably fossil seawater. The water has been modified in the reservoir by desiccation reactions related to the partial and local hydration of the igneous reservoir rock. The estimated reservoir temperature of 185 °C using three different calibrations of standard fluid geothermometers is in excellent agreement with measured and extrapolated temperatures in the borehole. The consistent application of different fluid geothermometers confirms the rock control of the fluid composition.

and kc370@cam.ac.uk 2 AUTHOR CONTRIBUTIONS KJC and JCRS designed the project. CCA and KJC developed the hydrogel technology. CL, CCA, BXT, MH, AW, CMV, and HTS performed the experiments and analysis. GGS analysed RNA sequencing data. WM performed the blastocyst injections. JCRS and KJC supervised ES cell experiments and analysis. PB supervised analysis of RNA sequencing data. KF supervised AFM analysis. CL and KJC wrote the paper. ACKNOWLEDGEMENTSWe are grateful to Peter Humphreys for assistance with imaging and imaging analysis, Sally Lees and staff for tissue culture work, Maike Paramor and staff for preparation of sequencing libraries, Michael A. Barber for alignment of sequencing data, G. Chu and staff for animal husbandry, CarlaMulas for comments on the manuscript, Ivan B. Dimov and Amelia Joy Thompson for help with AFM measurements. Rosa26-CreERT2+/+ cells were a kind gift from the lab of Bon-Kyoung Koo. Illumina RNA sequencing was performed at the CIGC, Cambridge. ABSTRACTStudies of mechanical signalling are typically performed by comparing cells cultured on soft and stiff hydrogel-based substrates. However, it is challenging to independently and robustly control both substrate stiffness and tethering of extracellular matrix (ECM) to substrates, making ECM tethering a potentially confounding variable in mechanical signalling investigations. Moreover, poor ECM tethering can lead to weak cell attachment. To address this, we developed StemBond hydrogels, a hydrogel formulation in which ECM tethering is stable and can be varied independently of stiffness.We show that soft StemBond hydrogels provide an optimal format for culturing embryonic stem (ES) cells. We find that soft StemBond substrates improve the homogeneity of ES cell populations, boost their self-renewal, and increase the efficiency of cellular reprogramming. Our findings underline how soft microenvironments impact mechanosensitive signalling pathways regulating self-renewal and differentiation, indicating that optimising the complete mechanical microenvironment will offer greater control over stem cell fate specification.

In many leukemia patients, a poor prognosis is attributed either to the development of chemotherapy resistance by leukemic stem cells (LSCs) or to the inefficient engraftment of transplanted hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM). Here, we build a 3D in vitro model system of bone marrow organoids (BMOs) that recapitulate several structural and cellular components of native BM. These organoids are formed in a high-throughput manner from the aggregation of endothelial and mesenchymal cells within hydrogel microwells. Accordingly, the mesenchymal compartment shows partial maintenance of its self-renewal and multilineage potential, while endothelial cells self-organize into an interconnected vessel-like network. Intriguingly, such a vascular compartment enhances the recruitment of HSPCs in a chemokine ligand/receptor-dependent manner, reminiscent of HSPC homing behavior in vivo. Additionally, we also model LSC migration and nesting in BMOs, thus highlighting the potential of this system as a well accessible and scalable preclinical model for candidate drug screening and patient-specific assays.