Development of bioactive hydrogel capsules for the 3D expansion of pluripotent stem cells in bioreactors

Pluripotent stem cells hold great promise for many pharmaceutical and therapeutic applications. However, the lack of scalable methodologies to expand these cells to clinically relevant numbers is a major roadblock in realizing their full potential. To address this problem, we report here a scalable approach for the expansion of pluripotent stem cells within bioactive hydrogel capsules in stirred bioreactors. To achieve rapid crosslinking of cellular microenvironments with tuneable, cell-instructive functionality, we combined calcium-mediated alginate (CaAlg) complexation with crosslinking of poly-(ethylene glycol) (PEG) macromers via a Michael-type addition. The resulting hybrid networks have been shown to have very good handling properties and can be readily decorated with biologically active signals such as integrin ligands or Cadherin-based motifs to influence the fate of mouse induced pluripotent stem (iPS) cells. Air-driven co-axial extrusion was used to reproducibly generate gel microcapsules in high-throughput. Furthermore, the gel capsules can be enveloped in a poly(L-lysine) shell to control swelling or molecular permeability independently of the gel composition. iPS cells entrapped within such capsules expanded with limited commitment to the endodermal lineage. Functionalization of gels with an appropriate density of Arg-Gly-Asp (RGD) ligands further increased the iPS cell expansion rate and reduced the spontaneous differentiation. Therefore, the combination of micro-scale instruction of cell fate by an engineered microenvironment and macro-scale cell manipulation in bioreactors opens up exciting opportunities for stem cell-based applications.

Quantitative analysis of SOX2 and OCT4 expression in pluripotency and differentiation

Daniel Strebinger

Embryonic stem (ES) cells have the capacity to give rise to all cell types of the adult organism and can be used as a model to study early cell fate decisions as they closely recapitulate in vivo events. The M and G1 phases have been suggested to be the key time windows for pluripotent cells to engage toward differentiation and SOX2 and OCT4 have been shown to orchestrate the dissolution of pluripotency and induction of cell fate commitment. Furthermore, it is known that the levels of some pluripotency transcription factors fluctuate over time, leading to the notion that ES cells experience dynamic heterogeneity, where for example low levels of NANOG can be found in cells prone to differentiation, whereas cells with high NANOG expression are in a robust pluripotent state. However, it remains unclear if and how (i) the action of transcription factors in specific cell cycle phases and (ii) protein level variations of factors that show mild differences between single cells impact cell fate decisions. This is in part due to the lack of quantitative single-cell meth-ods allowing dissecting the origin and functional consequences of gene expression heterogeneity. In this work, we established a method based on internal tagging of endogenous proteins with an excisable reporter to perform absolute molecule quantification in single living cells. This enabled us to investigate fluctuations of endogenous protein expression levels, the heterogeneity of degradation rates between individual cells and determine absolute amounts of proteins synthesized over the cell cycle. Additionally, inducible excision of the tag enabled us to estimate endogenous mRNA half-lives. Next, by using live-cell microscopy of fusion proteins, we show that SOX2 and OCT4 stay bound to mitotic chromosomes through their DNA binding domains. We then characterized the function of SOX2 at the M-G1 transition, showing that its absence specifically in this phase impairs pluripotency maintenance and abolishes its ability to induce neuroectodermal commitment. This suggests, that transcription factor activity at the M-G1 transition is essential for cell fate maintenance and differentiation. Lastly, we show that mild endogenous fluctuations in the levels of key transcription factors in ES cells bias cell fate decisions. We found that high OCT4 levels at the onset of differentiation increase the probability of single cells to commit to both neuroectoderm and mesendoderm. Further, we showed that high SOX2 levels result in a higher number of cells acquiring a neuroectodermal cell fate. This not only shows that the differentiation potential of embryonic stem cells is highly sensitive to small variations in intracellular concentrations of pluripotency transcription factors but further suggests that endogenous fluctuations of these are a major source of heterogeneity in cell fate decisions. In conclusion, the presented work identifies that the SOX2 activity in the M-G1 transition is important for cell fate maintenance and differentiation. Furthermore, the heterogeneity of protein levels governs cell fate decisions, suggesting the existence of different short-lived cell states in populations of presumed homogenous cells. We think that the minor differences in the levels of transcription factors and the cell cycle specific sensitivity of cells to transcription factor activity could be important mechanisms guiding maintenance and differentiation potential in various stem cell types.

EPFL2018

A role for mitotic bookmarking of SOX2 in pluripotency and differentiation

Aleksandr Benke, Andrea Callegari, Cédric Deluz, Torgil Elias Friman, Marion Leleu, Suliana Manley, Mahé Cécile Raccaud, Daniel Strebinger, David Michael Suter

Mitotic bookmarking transcription factors remain bound to chromosomes during mitosis and were proposed to regulate phenotypic maintenance of stem and progenitor cells at the mitosis-to-G1 (M-G1) transition. However, mitotic bookmarking remains largely unexplored in most stem cell types, and its functional relevance for cell fate decisions remains unclear. Here we screened for mitotic chromosome binding within the pluripotency network of embryonic stem (ES) cells and show that SOX2 and OCT4 remain bound to mitotic chromatin through their respective DNA-binding domains. Dynamic characterization using photobleaching-based methods and single-molecule imaging revealed quantitatively similar specific DNA interactions, but different nonspecific DNA interactions, of SOX2 and OCT4 with mitotic chromatin. Using ChIP-seq (chromatin immunoprecipitation [Chill combined with high-throughput sequencing) to assess the genome-wide distribution of SOX2 on mitotic chromatin, we demonstrate the bookmarking activity of SOX2 on a small set of genes. Finally, we investigated the function of SOX2 mitotic bookmarking in cell fate decisions and show that its absence at the M-G1 transition impairs pluripotency maintenance and abrogates its ability to induce neuroectodermal differentiation but does not affect reprogramming efficiency toward induced pluripotent stem cells. Our study demonstrates the mitotic bookmarking property of SOX2 and reveals its functional importance in pluripotency maintenance and ES cell differentiation.

Cold Spring Harbor Laboratory2016

Bioengineering stem cell niches in 2D and 3D

Nora Leonardi

Muscle degenerative disorders, such as Duchenne muscular dystrophy, have a profound impact on the quality of life and survival of patients. Transplantation of myoblasts to restore muscle function has been discouraging since these cells die and don't migrate but it has more recently been reported that muscle stem cells, known as satellite cells, have an extraordinary potential to contribute to muscle fiber regeneration, home to their niche and replenish the stem cell pool when injected freshly into damaged muscle. These cells however quickly lose their regenerative potential when cultured in vitro. Adult stem cells are known to be regulated by their microenvironment through a complex mixture of signals including soluble factors, matrix bound proteins, neural input and mechanical properties and the identification of extrinsic factors that can regulate muscle stem cell quiescence and self-renewal has been a focus of research in the Blau lab. Strikingly, recent studies in the Blau lab have shown that muscle stem cells cultured on compliant hydrogels coated with laminin were more viable than those cultured on tissue culture plastic and robustly contributed to muscle regeneration when transplanted into irradiated mouse leg muscles. The aim of this project was to follow up on these findings and to further elucidate the role biophysical and biochemical microenvironmental cues play in the regulation of muscle stem cell fate. Hydrogels have emerged as a popular material to recreate the stem cell niche in vitro due to their biocompatibility, high water content, tissue-like elasticity and diffusive properties and were employed here to fabricate substrates of varying rigidities. The physicochemical properties and tethered protein densities of a range of poyl(ethylene glycol) hydrogels were first characterized to define a suitable set of cell culture substrates. Gel stiffness could be linearly modulated from 2.5 to 45 kPa and the ligand density was found to be in the low femtomolar range. ELISA showed similar protein density on gels of different stiffnesses but immunofluorescence could not fully confirm this. Committed myoblasts were then seeded on these gels and shown to respond to substrate stiffness and ligand density in terms of morphology. Lastly, freshly isolated muscle stem cells were cultured on these gels and on rigid tissue culture plastic and shown to spread more, be more viable and grow more rapidly on stiffer gels as compared to soft ones. Myogenic progression was retrospectively analysed using immunohistochemistry but no differences between culture conditions were observed. Based on the influences of gel rigidity and ligand density observed in vitro, an in vivo assay, the ultimate test of stem cell function, is currently underway.