Lineage-Specific Mesenchymal Stromal Cells Derived from Human iPSCs Showed Distinct Patterns in Transcriptomic Profile and Extracellular Vesicle Production.

Over the past decades, mesenchymal stromal cells (MSCs) have been extensively investigated as a potential therapeutic cell source for the treatment of various disorders. Differentiation of MSCs from human induced pluripotent stem cells (iMSCs) has provided a scalable approach for the biomanufacturing of MSCs and related biological products. Although iMSCs shared typical MSC markers and functions as primary MSCs (pMSCs), there is a lack of lineage specificity in many iMSC differentiation protocols. Here, a stepwise hiPSC-to-iMSC differentiation method is employed via intermediate cell stages of neural crest and cytotrophoblast to generate lineage-specific MSCs with varying differentiation efficiencies and gene expression. Through a comprehensive comparison between early developmental cell types (hiPSCs, neural crest, and cytotrophoblast), two lineage-specific iMSCs, and six source-specific pMSCs, are able to not only distinguish the transcriptomic differences between MSCs and early developmental cells, but also determine the transcriptomic similarities of iMSC subtypes to postnatal or perinatal pMSCs. Additionally, it is demonstrated that different iMSC subtypes and priming conditions affected EV production, exosomal protein expression, and cytokine cargo.

IMSC-DERIVED EXTRACELLULAR VESICLES ATTENUATE LPS-INDUCED LUNG INJURY AND ENDOTOXEMIA IN MICE.

INTRODUCTION: We hypothesized extracellular vesicles (EVs) from preconditioned human induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) attenuate LPS-induced acute lung injury (ALI) and endotoxemia. METHODS: iMSCs were incubated with cell stimulation cocktail (CSC) and EVs were isolated. iMSC-EVs were characterized by size and EV markers. Bio-distribution of intratracheal (IT), intravenous and intraperitoneal injection of iMSC-EVs in mice was examined using IVIS. Uptake of iMSC-EVs in lung tissue, alveolar macrophages and RAW264.7 cells was also assessed. C57BL/6 mice were treated with IT/IP iMSC-EVs or vehicle ± IT/IP LPS to induce ALI/ARDS and endotoxemia. Lung tissues, plasma and BALF were harvested at 24 h. Lung histology, BALF neutrophil/macrophage, cytokine levels and total protein concentration were measured to assess ALI and inflammation. Survival studies were performed using IP LPS in mice for three days. RESULTS: iMSC-EV route of administration resulted in differential tissue distribution. iMSC-EVs were taken up by alveolar macrophages in mouse lung and cultured RAW264.7 cells. IT LPS-treated mice demonstrated marked histologic ALI, increased BALF neutrophils/macrophages and protein, increased BALF and plasma TNF-α/IL-6 levels. These parameters were attenuated by 2 h pre- or 2 h post-treatment with IT iMSC-EVs in ALI mice. Interestingly, the IT LPS-induced increase in IL-10 was augmented by iMSC-EVs. Mice treated with IP LPS showed increases in TNF-α and IL-6 that were downregulated by iMSC-EVs and LPS-induced mortality was ameliorated by iMSC-EVs. Administration of IT iMSC-EVs 2 h after LPS down-regulated the increase in pro-inflammatory cytokines (TNF-α/IL-6) by LPS and further increased IL-10 levels. CONCLUSIONS: iMSC-EVs attenuate the inflammatory effects of LPS on cytokine levels in ALI and IP LPS in mice. LPS-induced mortality was improved with administration of iMSC-EVs.

Controlling Mesenchyme Tissue Remodeling via Spatial Arrangement of Mechanical Constraints.

Tissue morphogenetic remodeling plays an important role in tissue repair and homeostasis and is often governed by mechanical stresses. In this study, we integrated an in vitro mesenchymal tissue experimental model with a volumetric contraction-based computational model to investigate how geometrical designs of tissue mechanical constraints affect the tissue remodeling processes. Both experimental data and simulation results verified that the standing posts resisted the bulk contraction of the tissues, leading to tissue thinning around the posts as gap extension and inward remodeling at the edges as tissue compaction. We changed the geometrical designs for the engineered mesenchymal tissues with different shapes of posts arrangements (triangle vs. square), different side lengths (6 mm vs. 8 mm), and insertion of a center post. Both experimental data and simulation results showed similar trends of tissue morphological changes of significant increase of gap extension and deflection compaction with larger tissues. Additionally, insertion of center post changed the mechanical stress distribution within the tissues and stabilized the tissue remodeling. This experimental-computational integrated model can be considered as a promising initiative for future mechanistic understanding of the relationship between mechanical design and tissue remodeling, which could possibly provide design rationale for tissue stability and manufacturing.

Remodeling of Architected Mesenchymal Microtissues Generated on Mechanical Metamaterials.

Mechanical metamaterials constitute a nascent category of architected structures comprising arranged periodic components with tailored geometrical features. These materials are now being employed as advanced medical implants due to their extraordinary mechanical properties over traditional devices. Nevertheless, to achieve desired tissue integration and regeneration, it is critical to study how the microarchitecture affects interactions between metamaterial scaffolds and living biological tissues. Based on human induced pluripotent stem cell technology and multiphoton lithography, we report the establishment of an in vitro microtissue model to study the integration and remodeling of human mesenchymal tissues on metamaterial scaffolds with different unit geometries. Microtissues showed distinct tissue morphologies and cellular behaviors between architected octet-truss and bowtie structures. Under the active force generated from mesenchymal tissues, the octet-truss and bowtie metamaterial scaffolds demonstrated unique instability phenomena, significantly different from uniform loading using conventional mechanical testing.

Engineering spatial-organized cardiac organoids for developmental toxicity testing.

Emerging technologies in stem cell engineering have produced sophisticated organoid platforms by controlling stem cell fate via biomaterial instructive cues. By micropatterning and differentiating human induced pluripotent stem cells (hiPSCs), we have engineered spatially organized cardiac organoids with contracting cardiomyocytes in the center surrounded by stromal cells distributed along the pattern perimeter. We investigated how geometric confinement directed the structural morphology and contractile functions of the cardiac organoids and tailored the pattern geometry to optimize organoid production. Using modern data-mining techniques, we found that pattern sizes significantly affected contraction functions, particularly in the parameters related to contraction duration and diastolic functions. We applied cardiac organoids generated from 600 µm diameter circles as a developmental toxicity screening assay and quantified the embryotoxic potential of nine pharmaceutical compounds. These cardiac organoids have potential use as an in vitro platform for studying organoid structure-function relationships, developmental processes, and drug-induced cardiac developmental toxicity.

Femtosecond laser induced densification within cell-laden hydrogels results in cellular alignment.

The unique capabilities of ultrafast lasers to introduce user-defined microscale modifications within 3D cell-laden hydrogels have been used to investigate fundamental cellular phenomenon such as adhesion, alignment, migration and organization. In this work, we report a new material modification phenomenon coined as 'densification' and its influence on the behavior of encapsulated cells. Femtosecond laser writing technique was used to write densified lines of width 1-5 µm within the bulk of gelatin methacrylate (GelMA) constructs. We found that densified micro-lines within cell-laden GelMA constructs resulted in preferential and localized alignment of encapsulated human endothelial cells. Degree of cellular alignment was characterized as a function of cell-culture time and the spacing between the densified line patterns. This phenomenon was found to be true for several cell lines, including mouse fibroblasts and osteocytes, and mesenchymal stem cells derived from human induced pluripotent cells. This first report of physical densification using fs lasers can be potentially extended for investigating cell behavior within other photosensitive hydrogels.

Serum-Free Manufacturing of Mesenchymal Stem Cell Tissue Rings Using Human-Induced Pluripotent Stem Cells.

Combination of stem cell technology and 3D biofabrication approaches provides physiological similarity to in vivo tissues and the capability of repairing and regenerating damaged human tissues. Mesenchymal stem cells (MSCs) have been widely used for regenerative medicine applications because of their immunosuppressive properties and multipotent potentials. To obtain large amount of high-quality MSCs without patient donation and invasive procedures, we differentiated MSCs from human-induced pluripotent stem cells (hiPSC-MSCs) using serum-free E6 media supplemented with only one growth factor (bFGF) and two small molecules (SB431542 and CHIR99021). The differentiated cells showed a high expression of common MSC-specific surface markers (CD90, CD73, CD105, CD106, CD146, and CD166) and a high potency for osteogenic and chondrogenic differentiation. With these cells, we have been able to manufacture MSC tissue rings with high consistency and robustness in pluronic-coated reusable PDMS devices. The MSC tissue rings were characterized based on inner diameter and outer ring diameter and observed cell-type-dependent tissue contraction induced by cell-matrix interaction. Our approach of simplified hiPSC-MSC differentiation, modular fabrication procedure, and serum-free culture conditions has a great potential for scalable manufacturing of MSC tissue rings for different regenerative medicine applications.