More Like this

1. Step-Wise Chondrogenesis of Human Induced Pluripotent Stem Cells and Purification Via a Reporter Allele Generated by CRISPR-Cas9 Genome Editing

   https://doi.org/10.1002/stem.2931  

   Adkar, Shaunak S. ; Wu, Chia-Lung ; Willard, Vincent P. ; Dicks, Amanda ; Ettyreddy, Adarsh ; Steward, Nancy ; Bhutani, Nidhi ; Gersbach, Charles A. ; Guilak, Farshid ( October 2018 , Stem Cells)

   The differentiation of human induced pluripotent stem cells (hiPSCs) to prescribed cell fates enables the engineering of patient-specific tissue types, such as hyaline cartilage, for applications in regenerative medicine, disease modeling, and drug screening. In many cases, however, these differentiation approaches are poorly controlled and generate heterogeneous cell populations. Here, we demonstrate cartilaginous matrix production in three unique hiPSC lines using a robust and reproducible differentiation protocol. To purify chondroprogenitors (CPs) produced by this protocol, we engineered a COL2A1-GFP knock-in reporter hiPSC line by CRISPR-Cas9 genome editing. Purified CPs demonstrated an improved chondrogenic capacity compared with unselected populations. The ability to enrich for CPs and generate homogenous matrix without contaminating cell types will be essential for regenerative and disease modeling applications. Stem Cells  2019;37:65–76

   Controlled differentiation of mesenchymal stem cells (MSCs) into the chondrogenic lineage is crucial for in vitro generation of neocartilage, yet achieving it remains challenging. Traditional protocols for MSC differentiation using exogenous inductive molecules, such as transforming growth factor-β, fall short in meeting the needs of clinical applications because they yield differentiated cells that exhibit hypertrophic characteristics and subsequently facilitate endochondral bone formation. The objective of the current study was to deliver endogenous inductive factors from juvenile articular chondrocytes to bone marrow-derived MSCs to drive MSC chondrogenic differentiation through cocultivation of the two cell types in the absence of direct physical contact and exogenous stimulators. An initial chondrocyte/MSC ratio of 63:1 was identified as the appropriate proportion of the two cell populations to ensure that coculture-driven MSC-differentiated (CDMD) cells replicated the cellular morphology, behavior, and phenotype of articular chondrocytes. In a three-dimensional agarose system, CDMD cells were further shown to develop into robust neocartilage structurally and mechanically stronger than chondrocyte-laden constructs and with reduced hypertrophic potential. Although MSCs tended to lose the ability to express CD44, an important regulator in cartilage biology, during the coculture induction, CDMD cells regained this function in the three-dimensional tissue cultivation. The present work establishes a chondrocyte/MSC coculture model that serves as a template to better understand chondrocyte-driven MSC differentiation and provides insights for improved strategies to develop clinically relevant cartilage tissue replacements.

    

   more » « less
2. Assembly of Human Stem Cell-Derived Cortical Spheroids and Vascular Spheroids to Model 3-D Brain-like Tissues

   https://doi.org/10.1038/s41598-019-42439-9  

   Song, Liqing ; Yuan, Xuegang ; Jones, Zachary ; Griffin, Kyle ; Zhou, Yi ; Ma, Teng ; Li, Yan ( April 2019 , Scientific Reports)

   Human cerebral organoids derived from induced pluripotent stem cells (iPSCs) provide novel tools for recapitulating the cytoarchitecture of human brain and for studying biological mechanisms of neurological disorders. However, the heterotypic interactions of neurovascular units, composed of neurons, pericytes, astrocytes, and brain microvascular endothelial cells, in brain-like tissues are less investigated. The objective of this study is to investigate the impacts of neural spheroids and vascular spheroids interactions on the regional brain-like tissue patterning in cortical spheroids derived from human iPSCs. Hybrid neurovascular spheroids were constructed by fusion of human iPSC-derived cortical neural progenitor cell (iNPC) spheroids, endothelial cell (iEC) spheroids, and the supporting human mesenchymal stem cells (MSCs). Single hybrid spheroids were constructed at different iNPC: iEC: MSC ratios of 4:2:0, 3:2:1 2:2:2, and 1:2:3 in low-attachment 96-well plates. The incorporation of MSCs upregulated the secretion levels of cytokines VEGF-A, PGE2, and TGF-β1 in hybrid spheroid system. In addition, tri-cultured spheroids had high levels of TBR1 (deep cortical layer VI) and Nkx2.1 (ventral cells), and matrix remodeling genes, MMP2 and MMP3, as well as Notch-1, indicating the crucial role of matrix remodeling and cell-cell communications on cortical spheroid and organoid patterning. Moreover, tri-culture system elevated blood-brain barrier gene expression (e.g., GLUT-1), CD31, and tight junction protein ZO1 expression. Treatment with AMD3100, a CXCR4 antagonist, showed the immobilization of MSCs during spheroid fusion, indicating a CXCR4-dependent manner of hMSC migration and homing. This forebrain-like model has potential applications in understanding heterotypic cell-cell interactions and novel drug screening in diseased human brain.

    

   more » « less
3. 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