Neural progenitor cells (NPCs) are a promising cell source to repair damaged nervous tissue. However, expansion of therapeutically relevant numbers of NPCs and their efficient differentiation into desired mature cell types remains a challenge. Material‐based strategies, including culture within 3D hydrogels, have the potential to overcome these current limitations. An ideal material would enable both NPC expansion and subsequent differentiation within a single platform. It has recently been demonstrated that cell‐mediated remodeling of 3D hydrogels is necessary to maintain the stem cell phenotype of NPCs during expansion, but the role of matrix remodeling on NPC differentiation and maturation remains unknown. By culturing NPCs within engineered protein hydrogels susceptible to degradation by NPC‐secreted proteases, it is identified that a critical amount of remodeling is necessary to enable NPC differentiation, even in highly degradable gels. Chemical induction of differentiation after sufficient remodeling time results in differentiation into astrocytes and neurotransmitter‐responsive neurons. Matrix remodeling modulates expression of the transcriptional co‐activator Yes‐associated protein, which drives expression of NPC stemness factors and maintains NPC differentiation capacity, in a cadherin‐dependent manner. Thus, cell‐remodelable hydrogels are an attractive platform to enable expansion of NPCs followed by differentiation of the cells into mature phenotypes for therapeutic use.
Neural progenitor cells (NPCs) are promising therapeutic candidates for nervous system regeneration. Significant efforts focus on developing hydrogel‐based approaches to facilitate the clinical translation of NPCs, from scalable platforms for stem cell production to injectable carriers for cell transplantation. However, fundamental questions surrounding NPC‐hydrogel interactions remain unanswered. While matrix degradability is known to regulate the stemness and differentiation capacity of NPCs, how degradability impacts NPC epigenetic regulation and secretory phenotype remains unknown. To address this question, NPCs encapsulated in recombinant protein hydrogels with tunable degradability are assayed for changes in chromatin organization and neurotrophin expression. In high degradability gels, NPCs maintain expression of stem cell factors, proliferate, and have large nuclei with elevated levels of the stemness‐associated activating histone mark H3K4me3. In contrast, NPCs in low degradability gels exhibit more compact, rounded nuclei with peripherally localized heterochromatin, are non‐proliferative yet non‐senescent, and maintain expression of neurotrophic factors with potential therapeutic relevance. This work suggests that tuning matrix degradability may be useful to direct NPCs toward either a more‐proliferative, stem‐like phenotype for cell replacement therapies, or a more quiescent‐like, pro‐secretory phenotype for soluble factor‐mediated therapies.
more » « less- NSF-PAR ID:
- 10456634
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Healthcare Materials
- Volume:
- 9
- Issue:
- 18
- ISSN:
- 2192-2640
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract -
Synthetic matrices that are cytocompatible, cell adhesive and cell responsive are needed for the engineering of implantable, secretory salivary gland constructs to treat radiation induced xerostomia or dry mouth. Here, taking advantage of the bioorthogonality of the Michael-type addition reaction, hydrogels with comparable stiffness but varying degrees of degradability (100% degradable: 100DEG; 50% degradable: 50DEG; and non-degradable: 0DEG) by cell-secreted matrix metalloproteases (MMPs) were synthesized using thiolated HA (HA-SH), maleimide (MI)-conjugated integrin-binding peptide (RGD-MI) and MI-functionalized peptide crosslinkers that are protease degradable (GIW-bisMI) or non-degradable (GIQ-bisMI). Organized multicellular structures developed readily in all hydrogels from dispersed primary human salivary gland stem/progenitor cells (hS/PCs). As the matrix became progressively degradable, cells proliferated more readily and the multicellular structures became larger, less spherical, and more lobular. Immunocytochemical analysis showed positive staining for stem/progenitor cell markers CD44 and keratin 5 (K5) in all three types of cultures, and positive staining for the acinar marker α-amylase under 50DEG and 100DEG conditions. Quantitatively at the mRNA level, the expression levels of key stem/progenitor markers KIT, KRT5, and ETV4/5 were significantly increased in the degradable gels as compared to the non-degradable counterparts. Western blot analyses revealed that imparting matrix degradation led to >3.8-fold increase in KIT expression by day 15. The MMP-degradable hydrogels also promoted the development of a secretary phenotype, as evidenced by the upregulation of acinar markers α-amylase (AMY), aquaporin-5 (AQP5), and sodium-potassium-chloride cotransporter 1 (SLC12A2). Collectively, we show that cell-mediated matrix remodeling is necessary for the development of regenerative pro-acinar progenitor cells from hS/PCs.more » « less
-
Osteoarthritis (OA), a chronic and degenerative joint disease, remains a challenge in treatment due to the lack of disease-modifying therapies. As a promising therapeutic agent, adipose-derived stem cells (ADSCs) have an effective anti-inflammatory and chondroprotective paracrine effect that can be enhanced by genetic modification. Unfortunately, direct cell delivery without matrix support often results in poor viability of therapeutic cells. Herein, a hydrogel implant approach that enabled intra-articular delivery of gene-engineered ADSCs was developed for improved therapeutic outcomes in a surgically induced rat OA model. An injectable extracellular matrix (ECM)-mimicking hydrogel was prepared as the carrier for cell delivery, providing a favorable microenvironment for ADSC spreading and proliferation. The ECM-mimicking hydrogel could reduce cell death during and post injection. Additionally, ADSCs were genetically modified to overexpress transforming growth factor-β1 (TGF-β1), one of the paracrine factors that exert an anti-inflammatory and pro-anabolic effect. The gene-engineered ADSCs overexpressing TGF-β1 (T-ADSCs) had an enhanced paracrine effect on OA-like chondrocytes, which effectively decreased the expression of tumor necrosis factor-alpha and increased the expression of collagen II and aggrecan. In a surgically induced rat OA model, intra-articular injection of the T-ADSC-loaded hydrogel markedly reduced cartilage degeneration, joint inflammation, and the loss of the subchondral bone. Taken together, this study provides a potential biomaterial strategy for enhanced OA treatment by delivering the gene-engineered ADSCs within an ECM-mimicking hydrogel.more » « less
-
more » « less
Abstract Microporous annealed particle (MAP) scaffolds are generated from assembled hydrogel microparticles (microgels). It has been previously demonstrated that MAP scaffold are porous, biocompatible, and recruit neural progenitor cells (NPCs) to the stroke cavity after injection into the stroke core. Here, the goal is to study NPC fate inside MAP scaffolds in vitro. To create plain microgels that can later be converted to contain different types of bioactivities, the inverse electron‐demand Diels–Alder reaction between tetrazine and norbornene is utilized, which allows the post‐modification of plain microgels stoichiometrically. As a result of adhesive peptide attachment, NPC spreading leads to contractile force generation which can be recorded by tracking microgel displacement. Alternatively, non‐adhesive peptide integration results in neurosphere formation that grows within the void space of MAP scaffolds. Although the formed neurospheres do not impose a contractile force on the scaffolds, they are seen to continuously transverse the scaffolds. It is concluded that MAP scaffolds can be engineered to either promote neurogenesis or enhance stemness depending on the chosen post‐modifications of the microgels, which can be key in modulating their phenotypes in various applications in vivo.
-
Age is a leading risk factor for developing breast cancer. This may be in part to the time required for acquiring sufficient cancer mutations; however, stromal cells that accumulate in tissues and undergo senescence eventually develop a senescence-associated secretory phenotype that alters the microenvironment to promote cancer. Our focus is on mesenchymal stem cells (MSCs) – stromal cells recruited to tumors due to their natural tropism for inflammatory tissues; MSCs have been shown to enhance the metastatic potential of tumor cells through direct interactions or paracrine signaling within the tumor. In the tumor, MSCs can differentiate into carcinoma-associated fibroblasts that play a central role in tumor growth and matrix remodeling. We recently investigated the molecular and mechanical differences in pre- and post- senescent MSCs and how their interactions with MDA-MB-231 breast cancer cells contribute to malignancy. Our data show post-senescent MSCs are larger and less motile, with more homogeneous mechanical properties than pre-senescent MSCs. In-depth omics analysis revealed differentially regulated genes and peptides including factors related to inflammatory cytokines, cell adhesion to the extracellular matrix, and cytoskeletal regulation. A 3D co-culture model was used to assess the effects of pre- and post- senescent MSCs on collagen matrix remodeling. Although post-senescent MSCs were far less motile than pre-senescent MSCs and less contractile with the matrix, they profoundly altered matrix protein deposition and crosslinking, which resulted in local matrix stiffening effects. Post-senescent MSCs also induced an invasive breast cancer cell phenotype, characterized by increased proliferation and invasion of breast cancer cells. This invasive breast cancer cell behavior was further amplified when MDA-MB-231 was co-cultured with a mixture of pre- and post- senescent MSCs; this result was attributed to matrix remodeling and soluble factor secretion effects of post-senescent MSCs, which enhanced the migration of pre-senescent MSCs allowing them to form tracks in the collagen network for cancer cells to follow. Finally, molecular inhibitors targeting actomyosin contractility and adhesion were used to alter MSC interactions with breast cancer cells. Actin depolymerizing agent and focal adhesion kinase inhibitor were most efficient and completely able to block the effects of post-senescent MSCs on MDA-MB-231 invasion in collagen gels. This comprehensive approach can be used to identify molecular pathways regulating heterotypic interactions of post-senescent MSCs with other cells in the tumor. Furthermore, the local matrix stiffening effect of post-senescent MSCs may play a critical role in breast cancer progression.more » « less
