Marina Prewitz

Tightly anchored tissue-mimetic matrices as instructive stem cell microenvironments

A major obstacle in defining the exact role of extracellular matrix (ECM) in stem cell niches is the lack of suitable in vitro methods that recapitulate complex ECM microenvironments. Here we describe a methodology that permits reliable anchorage of native cell–secreted ECM to culture carriers. We validated our approach by fabricating two types of human bone marrow–specific ECM substrates that were robust enough to support human mesenchymal stem cells (MSCs) and hematopoietic stem and progenitor cells in vitro. We characterized the molecular composition, structural features and nanomechanical properties of the MSC-derived ECM preparations and demonstrated their ability to support expansion and differentiation of bone marrow stem cells. Our methodology enables the deciphering and modulation of native-like multicomponent ECMs of tissue-resident stem cells and will therefore prepare the ground for a more rational design of engineered stem cell niches.

Matrix elasticity regulates the secretory profile of human bone marrow-derived multipotent mesenchymal stromal cells (MSCs)

The therapeutic efficacy of multipotent mesenchymal stromal cells (MSCs) is attributed to particular MSC-derived cytokines and growth factors. As MSCs are applied locally to target organs or home there after systemic administration, they experience diverse microenvironments that are biochemically and biophysically distinct. Here we use well-defined in vitro conditions to study the impact of substrate elasticity on MSC-derived trophic factors. By varying hydrogel compliance, the elasticity of brain and muscle tissue was mimicked. We screened >90 secreted factors at the protein level, finding a diverse elasticity-dependent expression pattern. In particular, IL-8 was up-regulated as much as 90-fold in MSCs cultured for 2days on hard substrates, whereas levels were consistently low on soft substrates. In summary, we show substrate elasticity directly affects MSC paracrine expression, a relevant finding for therapies administering MSCs in vivo.

Polymeric biomaterials for stem cell bioengineering

This review covers the application of polymeric materials in stem cell bioengineering. Main emphasis is directed towards current material design concepts that mimic distinct exogenous signals of the stem cell microenvironment. Progress within the field of stem cell-specific biomaterials will be discussed, focusing on pluripotent, hematopoietic, mesenchymal and neural stem cells. The future role of biomaterials will be outlined with possible applications for cell reprogramming and engineering cancer cell microenvironments.

Knockdown of the co-chaperone Hop promotes extranuclear accumulation of Stat3 in mouse embryonic stem cells

A key event in the mechanism of mouse embryonic stem cell (mESC) pluripotency is phosphorylation, dimerisation and translocation to the nucleus of the signal transducer and activator of transcription3, Stat3. We used RNAi to suppress the levels of the co-chaperone Hsp70/Hsp90 organising protein (Hop) in an mESC line. Hop knockdown caused 68% depletion in Stat3 mRNA levels, decreased soluble pYStat3 levels, and led to an extranuclear accumulation of Stat3. The major binding partner of Hop, Hsp90, co-localised with a small non-nuclear fraction of Stat3 in mESCs, and both Stat3 and Hop co-precipitated with Hsp90. Hop knockdown did not affect Nanog and Oct4 protein levels; however, Nanog mRNA levels were decreased. We found that in the absence of Hop, mESCs lost their pluripotent ability to form embryoid bodies with a basement membrane. These data suggest that Hop facilitates the phosphorylation and nuclear translocation of Stat3, implying a role for the Hsp70/Hsp90 chaperone heterocomplex machinery in pluripotency signalling.

Extracellular matrix deposition of bone marrow stroma enhanced by macromolecular crowding

Decellularized extracellular matrices (ECM) from in vitro cell cultures can serve as in vivo-like matrix scaffolds for modulating cell-ECM interactions. Macromolecular crowding (MMC), the supplementation of synthetic or naturally occurring molecules resulting in excluded volume effects (EVE), has been demonstrated to provide valuable options for recapitulating the physiological environment of cells during matrix secretion. Human mesenchymal stem cell (MSC)-derived ECM was produced upon supplementation of standard culture medium with three different macromolecules of various size (10-500 kDa). Matrix secretion, ECM morphology and composition were compared for matrices obtained from crowded and non-crowded MSC cultures. In the context of generating functional stem cell niches, the MSC-derived bone marrow mimetic ECM scaffolds were tested for their supportive effect to maintain and expand human hematopoietic stem and progenitor cells (HSPC) in vitro. MMC in combination with metabolic stimulation of MSC was found to result in tissue-specific, highly organized ECM capable of retaining glycosaminoglycans and growth factors to effectively build in vitro microenvironments that support HSPC expansion.

In Vitro Model of Metastasis to Bone Marrow Mediates Prostate Cancer Castration Resistant Growth through Paracrine and Extracellular Matrix Factors

The spread of prostate cancer cells to the bone marrow microenvironment and castration resistant growth are key steps in disease progression and significant sources of morbidity. However, the biological significance of mesenchymal stem cells (MSCs) and bone marrow derived extracellular matrix (BM-ECM) in this process is not fully understood. We therefore established an in vitro engineered bone marrow tissue model that incorporates hMSCs and BM-ECM to facilitate mechanistic studies of prostate cancer cell survival in androgen-depleted media in response to paracrine factors and BM-ECM. hMSC-derived paracrine factors increased LNCaP cell survival, which was in part attributed to IGFR and IL6 signaling. In addition, BM-ECM increased LNCaP and MDA-PCa-2b cell survival in androgen-depleted conditions, and induced chemoresistance and morphological changes in LNCaPs. To determine the effect of BM-ECM on cell signaling, the phosphorylation status of 46 kinases was examined. Increases in the phosphorylation of MAPK pathway-related proteins as well as sustained Akt phosphorylation were observed in BM-ECM cultures when compared to cultures grown on plasma-treated polystyrene. Blocking MEK1/2 or the PI3K pathway led to a significant reduction in LNCaP survival when cultured on BM-ECM in androgen-depleted conditions. The clinical relevance of these observations was determined by analyzing Erk phosphorylation in human bone metastatic prostate cancer versus non-metastatic prostate cancer, and increased phosphorylation was seen in the metastatic samples. Here we describe an engineered bone marrow model that mimics many features observed in patients and provides a platform for mechanistic in vitro studies.

Macroporous biohybrid cryogels for co-housing pancreatic islets with mesenchymal stromal cells.

UNLABELLED Intrahepatic transplantation of allogeneic pancreatic islets offers a promising therapy for type 1 diabetes. However, long-term insulin independency is often not achieved due to severe islet loss shortly after transplantation. To improve islet survival and function, extrahepatic biomaterial-assisted transplantation of pancreatic islets to alternative sites has been suggested. Herein, we present macroporous, star-shaped poly(ethylene glycol) (starPEG)-heparin cryogel scaffolds, covalently modified with adhesion peptides, for the housing of pancreatic islets in three-dimensional (3D) co-culture with adherent mesenchymal stromal cells (MSC) as accessory cells. The implantable biohybrid scaffolds provide efficient transport properties, mechanical protection, and a supportive extracellular environment as a desirable niche for the islets. MSC colonized the cryogel scaffolds and produced extracellular matrix proteins that are important components of the natural islet microenvironment known to facilitate matrix-cell interactions and to prevent cellular stress. Islets survived the seeding procedure into the cryogel scaffolds and secreted insulin after glucose stimulation in vitro. In a rodent model, intact islets and MSC could be visualized within the scaffolds seven days after subcutaneous transplantation. Overall, this demonstrates the potential of customized macroporous starPEG-heparin cryogel scaffolds in combination with MSC to serve as a multifunctional islet supportive carrier for transplantation applications. STATEMENT OF SIGNIFICANCE Diabetes results in the insufficient production of insulin by the pancreatic β-cells in the islets of Langerhans. Transplantation of pancreatic islets offers valuable options for treating the disease; however, many transplanted islets often do not survive the transplantation or die shortly thereafter. Co-transplanted, supporting cells and biomaterials can be instrumental for improving islet survival, function and protection from the immune system. In the present study, islet supportive hydrogel sponges were explored for the co-transplantation of islets and mesenchymal stromal cells. Survival and continued function of the supported islets were demonstrated in vitro. The in vivo feasibility of the approach was shown by transplantation in a mouse model.

Dewaxed ECM: A simple method for analyzing cell behaviour on decellularized extracellular matrices

Decellularization techniques have been used on a wide variety of tissues to create cell‐seedable scaffolds for tissue engineering. Finding a suitable decellularization protocol for a certain type of tissue can be laborious, especially when organ perfusion devices are needed. In this study, we report a quick and simple method for comparing decellularization protocols combining the use of paraffin slices and two‐dimensional cell cultures. We developed three decellularization protocols for adult murine kidney that yielded decellularized extracellular matrices (ECMs) with varying histological properties. The resulting paraffin‐embedded ECM slices were deparaffinized and reseeded with murine embryonic stem cells (mESCs). We analyzed cell attachment four days post seeding via determination of cell numbers, and used quantitative Real‐Time PCR 13 days post seeding to measure gene expression levels of two genes associated with renal development, Pax2 and Pou3f3. The three decellularization protocols produced kidney‐matrices that showed clearly distinguishable results. We demonstrated that formerly paraffin‐embedded decellularized ECMs can effectively influence differentiation of stem cells. This method can be used to identify optimal decellularization protocols for recellularization of three‐dimensional tissue‐scaffolds with embryonic stem cells and other tissue‐specific cell types. Copyright

Macromolecular crowding for tailoring tissue-derived fibrillated matrices

Tissue-derived fibrillated matrices can be instrumental for the in vitro reconstitution of multiphasic extracellular microenvironments. However, despite of several advantages, the obtained scaffolds so far offer a rather narrow range of materials characteristics only. In this work, we demonstrate how macromolecular crowding (MMC) - the supplementation of matrix reconstitution media with synthetic or natural macromolecules in ways to create excluded volume effects (EVE) - can be employed for tailoring important structural and biophysical characteristics of kidney-derived fibrillated matrices. Porcine kidneys were decellularized, ground and the obtained extracellular matrix (ECM) preparations were reconstituted under varied MMC conditions. We show that MMC strongly influences the fibrillogenesis kinetics and impacts the architecture and the elastic modulus of the reconstituted matrices, with diameters and relative alignment of fibrils increasing at elevated concentrations of the crowding agent Ficoll400, a nonionic synthetic polymer of sucrose. Furthermore, we demonstrate how MMC modulates the distribution of key ECM molecules within the reconstituted matrix scaffolds. As a proof of concept, we compared different variants of kidney-derived fibrillated matrices in cell culture experiments referring to specific requirements of kidney tissue engineering approaches. The results revealed that MMC-tailored matrices support the morphogenesis of human umbilical vein endothelial cells (HUVECs) into capillary networks and of murine kidney stem cells (KSCs) into highly branched aggregates. The established methodology is concluded to provide generally applicable new options for tailoring tissue-specific multiphasic matrices in vitro. STATEMENT OF SIGNIFICANCE Tissue-derived fibrillated matrices can be instrumental for the in vitro reconstitution of multiphasic extracellular microenvironments. However, despite of several advantages, the obtained scaffolds so far offer a rather narrow range of materials characteristics only. Using the kidney matrix as a model, we herein report a new approach for tailoring tissue-derived fibrillated matrices by means of macromolecular crowding (MMC), the supplementation of reconstitution media with synthetic or natural macromolecules. MMC-modulation of matrix reconstitution is demonstrated to allow for the adjustment of fibrillation kinetics and nano-architecture, fiber diameter, alignment, and matrix elasticity. Primary human umbilical vein endothelial cells (HUVEC) and murine kidney stem cells (KSC) were cultured within different variants of fibrillated kidney matrix scaffolds. The results showed that MMC-tailored matrices were superior in supporting desired morphogenesis phenomena of both cell types.

Polymere Biomaterialien als zelluläre Mikromilieus

Polymeric biomaterials can be instrumental for expanding and differentiating stem and progenitor cells in culture. Key to success of these approaches is the defined modulation of biomolecular and physical signals governing cellular microenvironments. Current concepts for effective stem cell culture carriers rely on cell-secreted decellularized matrices, reconstituted assemblies of biopolymers of extracellular matrices and on biohybrid or fully synthetic polymer hydrogels with bioactive units.