More Like this

1. Cyclical aggregation extends in vitro expansion potential of human mesenchymal stem cells

   https://doi.org/10.1038/s41598-020-77288-4  

   Bijonowski, Brent M. ; Yuan, Xuegang ; Jeske, Richard ; Li, Yan ; Grant, Samuel C. ( December 2020 , Scientific Reports)

   null (Ed.)

   Abstract Mesenchymal stem cell (MSC)-based therapy has shown great promises in various animal disease models. However, this therapeutic potency has not been well claimed when applied to human clinical trials. This is due to both the availability of MSCs at the time of administration and lack of viable expansion strategies. MSCs are very susceptible to in vitro culture environment and tend to adapt the microenvironment which could lead to cellular senescence and aging. Therefore, extended in vitro expansion induces loss of MSC functionality and its clinical relevance. To combat this effect, this work assessed a novel cyclical aggregation as a means of expanding MSCs to maintain stem cell functionality. The cyclical aggregation consists of an aggregation phase and an expansion phase by replating the dissociated MSC aggregates onto planar tissue culture surfaces. The results indicate that cyclical aggregation maintains proliferative capability, stem cell proteins, and clonogenicity, and prevents the acquisition of senescence. To determine why aggregation was responsible for this phenomenon, the integrated stress response pathway was probed with salubrial and GSK-2606414. Treatment with salubrial had no significant effect, while GSK-2606414 mitigated the effects of aggregation leading to in vitro aging. This method holds the potential to increase the clinical relevance of MSC therapeutic effects from small model systems (such as rats and mice) to humans, and may open the potential of patient-derived MSCs for treatment thereby removing the need for immunosuppression. 

   more » « less
2. Synthetic, Chemically Defined Polymer‐Coated Microcarriers for the Expansion of Human Mesenchymal Stem Cells

   https://doi.org/10.1002/mabi.201800299  

   Krutty, John D. ; Dias, Andrew D. ; Yun, Junsu ; Murphy, William L. ; Gopalan, Padma ( November 2018 , Macromolecular Bioscience)

   Mesenchymal stem cells (MSC), also called marrow stromal cells, are adult cells that have attracted interest for their potential uses in therapeutic applications. There is a pressing need for scalable culture systems due to the large number of cells needed for clinical treatments. Here, a tailorable thin polymer coating—poly(poly(ethylene glycol) methyl ether methacrylate‐ran‐vinyl dimethyl azlactone‐ran‐glycidyl methacrylate) [P(PEGMEMA‐r‐VDM‐r‐GMA); PVG]—to the surface of commercially available polystyrene and glass microcarriers to create chemically defined surfaces for large‐scale cell expansion is applied. These chemically defined microcarriers create a reproducible surface that does not rely on the adsorption of xenogenic serum proteins to mediate cell adhesion. Specifically, this coating method anchors PVG copolymer through ring opening nucleophilic attack by amine residues on poly‐l‐lysine that is pre‐adsorbed to the surface of microcarriers. Importantly, this anchoring reaction preserves the monomer VDM reactivity for subsequent functionalization with an integrin‐specific Arg‐Gly‐Asp peptide to enable cell adhesion and expansion via a one‐step reaction in aqueous media. MSCs cultured on PVG‐coated microcarriers achieve sixfold expansion—similar to the expansion achieved on PS microcarriers—and retain their ability to differentiate after harvesting.

    

   more » « less
3. Soluble Signals and Remodeling in a Synthetic Gelatin‐Based Hematopoietic Stem Cell Niche

   https://doi.org/10.1002/adhm.201900751  

   Gilchrist, Aidan E. ; Lee, Sunho ; Hu, Yuhang ; Harley, Brendan A. C. ( September 2019 , Advanced Healthcare Materials)

   Hematopoietic stem cells (HSCs) reside in the bone marrow within niches that provide microenvironmental signals in the form of biophysical cues, bound and diffusible biomolecules, and heterotypic cell–cell interactions that influence HSC fate decisions. This study seeks to inform the development of a synthetic culture platform that promotes ex vivo HSC expansion without exhaustion. A library of methacrylamide‐functionalized gelatin (GelMA) hydrogels is used to explore remodeling and crosstalk from mesenchymal stromal cells (MSCs) on the expansion and quiescence of murine HSCs. The use of a degradable GelMA hydrogel enables MSC‐mediated remodeling, yielding dynamic shifts in the matrix environment over time. An initially low‐diffusivity hydrogel for co‐culture of hematopoietic stem and progenitor cells to MSCs facilitates maintenance of an early progenitor cell population over 7 days. Excitingly, this platform promotes retention of a quiescent HSC population compared to HSC monocultures. These studies reveal MSC‐density–dependent upregulation of MMP‐9 and changes in hydrogel mechanical properties (ΔE= 2.61 ± 0.72) suggesting MSC‐mediated matrix remodeling may contribute to a dynamic culture environment. Herein, a 3D hydrogel is reported for ex vivo HSC culture, in which HSC expansion and quiescence is sensitive to hydrogel properties, MSC co‐culture, and MSC‐mediated hydrogel remodeling.

    

   more » « less
4. Mesenchymal Stem Cell Culture within Perfusion Bioreactors Incorporating 3D‐Printed Scaffolds Enables Improved Extracellular Vesicle Yield with Preserved Bioactivity

   https://doi.org/10.1002/adhm.202300584  

   Kronstadt, Stephanie M. ; Patel, Divya B. ; Born, Louis J. ; Levy, Daniel ; Lerman, Max J. ; Mahadik, Bhushan ; McLoughlin, Shannon T. ; Fasuyi, Arafat ; Fowlkes, Lauren ; Van Heyningen, Lauren Hoorens ; et al ( April 2023 , Advanced Healthcare Materials)

   Extracellular vesicles (EVs) are implicated as promising therapeutics and drug delivery vehicles in various diseases. However, successful clinical translation will depend on the development of scalable biomanufacturing approaches, especially due to the documented low levels of intrinsic EV‐associated cargo that may necessitate repeated doses to achieve clinical benefit in certain applications. Thus, here the effects of a 3D‐printed scaffold‐perfusion bioreactor system are assessed on the production and bioactivity of EVs secreted from bone marrow‐derived mesenchymal stem cells (MSCs), a cell type widely implicated in generating EVs with therapeutic potential. The results indicate that perfusion bioreactor culture induces an ≈40‐80‐fold increase (depending on measurement method) in MSC EV production compared to conventional cell culture. Additionally, MSC EVs generated using the perfusion bioreactor system significantly improve wound healing in a diabetic mouse model, with increased CD31⁺staining in wound bed tissue compared to animals treated with flask cell culture‐generated MSC EVs. Overall, this study establishes a promising solution to a major EV translational bottleneck, with the capacity for tunability for specific applications and general improvement alongside advancements in 3D‐printing technologies.

    

   more » « less
5. Aggregation‐induced integrated stress response rejuvenates culture‐expanded human mesenchymal stem cells

   https://doi.org/10.1002/bit.27474  

   Bijonowski, Brent M. ; Fu, Qin ; Yuan, Xuegang ; Irianto, Jerome ; Li, Yan ; Grant, Samuel C. ; Ma, Teng ( July 2020 , Biotechnology and Bioengineering)

   Protein homeostasis is critical for cellular function, as loss of homeostasis is attributed to aging and the accumulation of unwanted proteins. Human mesenchymal stem cells (MSCs) have shown promising therapeutic potential due to their impressive abilities to secrete inflammatory modulators, angiogenic, and regenerative cytokines. However, there exists the problem of human MSC expansion with compromised therapeutic quality. Duringin vitro expansion, human MSCs are plated on stiff plastics and undergo culture adaptation, which results in aberrant proliferation, shifts in metabolism, and decreased autophagic activity. It has previously been shown that three‐dimensional (3D) aggregation can reverse some of these alterations by heightening autophagy and recovering the metabolic state back to a naïve phenotype. To further understand the proteostasis in human MSC culture, this study investigated the effects of 3D aggregation on the human MSC proteome to determine the specific pathways altered by aggregation. The 3D aggregates and 2D cultures of human MSCs derived from bone marrow (bMSC) and adipose tissue (ASC) were analyzed along with differentiated human dermal fibroblasts (FB). The proteomics analysis showed the elevated eukaryotic initiation factor 2 pathway and the upregulated activity of the integrated stress response (ISR) in 3D aggregates. Specific protein quantification further determined that bMSC and ASC responded to ISR, while FB did not. 3D aggregation significantly increased the ischemic survival of bMSCs and ASCs. Perturbation of ISR with small molecules salubrinal and GSK2606414 resulted in differential responses of bMSC, ASC, and FB. This study indicates that aggregation‐based preconditioning culture holds the potential for improving the therapeutic efficacy of expanded human MSCs via the establishment of ISR and homeostasis.

    

   more » « less