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.
The objective of this work is to investigate the effect of devitalized human mesenchymal stem cells (hMSCs) and endothelial colony‐forming cells (ECFCs) seeded on mineralized nanofiber microsheets on protein release, osteogenesis, vasculogenesis, and macrophage polarization. Calcium phosphate nanocrystals are grown on the surface of aligned, functionalized nanofiber microsheets. The microsheets are seeded with hMSCs, ECFCs, or a mixture of hMSCs + ECFCs, cultured for cell attachment, differentiated to the osteogenic or vasculogenic lineage, and devitalized by lyophilization. The release kinetic of total protein, bone morphogenetic protein‐2 (BMP2), and vascular endothelial growth factor (VEGF) from the devitalized microsheets is measured. Next, hMSCs and/or ECFCs are seeded on the devitalized cell microsheets and cultured in the absence of osteo‐/vasculoinductive factors to determine the effect of devitalized cell microsheets on hMSC/ECFC differentiation. Human macrophages are seeded on the microsheets to determine the effect of devitalized cells on macrophage polarization. Based on the results, devitalized undifferentiated hMSC and vasculogenic‐differentiated ECFC microsheets have highest sustained release of BMP2 and VEGF, respectively. The devitalized hMSC microsheets do not affect M2 macrophage polarization while vascular‐differentiated, devitalized ECFC microsheets do not affect M1 polarization. Both groups stimulate higher M2 macrophage polarization compared to M1.
more » « less- NSF-PAR ID:
- 10034625
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Biosystems
- Volume:
- 1
- Issue:
- 3
- ISSN:
- 2366-7478
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Implantation of stem cells for tissue regeneration faces significant challenges such as immune rejection and teratoma formation. Cell‐free tissue regeneration thus has a potential to avoid these problems. Stem cell derived exosomes do not cause immune rejection or generate malignant tumors. Here, exosomes that can induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) are identified and used to decorate 3D‐printed titanium alloy scaffolds to achieve cell‐free bone regeneration. Specifically, the exosomes secreted by hMSCs osteogenically pre‐differentiated for different times are used to induce the osteogenesis of hMSCs. It is discovered that pre‐differentiation for 10 and 15 days leads to the production of osteogenic exosomes. The purified exosomes are then loaded into the scaffolds. It is found that the cell‐free exosome‐coated scaffolds regenerate bone tissue as efficiently as hMSC‐seeded exosome‐free scaffolds within 12 weeks. RNA‐sequencing suggests that the osteogenic exosomes induce the osteogenic differentiation by using their cargos, including upregulated osteogenic miRNAs (Hsa‐miR‐146a‐5p, Hsa‐miR‐503‐5p, Hsa‐miR‐483‐3p, and Hsa‐miR‐129‐5p) or downregulated anti‐osteogenic miRNAs (Hsa‐miR‐32‐5p, Hsa‐miR‐133a‐3p, and Hsa‐miR‐204‐5p), to activate the PI3K/Akt and MAPK signaling pathways. Consequently, identification of osteogenic exosomes secreted by pre‐differentiated stem cells and the use of them to replace stem cells represent a novel cell‐free bone regeneration strategy.
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Abstract A new approach is described for fabricating 3D poly(
ε ‐caprolactone) (PCL)/gelatin (1:1) nanofiber aerogels with patterned macrochannels and anisotropic microchannels by freeze‐casting with 3D‐printed sacrificial templates. Single layer or multiple layers of macrochannels are formed through an inverse replica of 3D‐printed templates. Aligned microchannels formed by partially anisotropic freezing act as interconnected pores between templated macrochannels. The resulting macro‐/microchannels within nanofiber aerogels significantly increase preosteoblast infiltration in vitro. The conjugation of vascular endothelial growth factor (VEGF)‐mimicking QK peptide to PCL/gelatin/gelatin methacryloyl (1:0.5:0.5) nanofiber aerogels with patterned macrochannels promotes the formation of a microvascular network of seeded human microvascular endothelial cells. Moreover, nanofiber aerogels with patterned macrochannels and anisotropic microchannels show significantly enhanced cellular infiltration rates and host tissue integration compared to aerogels without macrochannels following subcutaneous implantation in rats. Taken together, this novel class of nanofiber aerogels holds great potential in biomedical applications including tissue repair and regeneration, wound healing, and 3D tissue/disease modeling. -
null (Ed.)Background Volumetric tissue-engineered constructs are limited in development due to the dependence on well-formed vascular networks. Scaffold pore size and the mechanical properties of the matrix dictates cell attachment, proliferation and successive tissue morphogenesis. We hypothesize scaffold pore architecture also controls stromal-vessel interactions during morphogenesis. Methods The interaction between mesenchymal stem cells (MSCs) seeded on hydroxyapatite scaffolds of 450, 340, and 250 μm pores and microvascular fragments (MVFs) seeded within 20 mg/mL fibrin hydrogels that were cast into the cell-seeded scaffolds, was assessed in vitro over 21 days and compared to the fibrin hydrogels without scaffold but containing both MSCs and MVFs. mRNA sequencing was performed across all groups and a computational mechanics model was developed to validate architecture effects on predicting vascularization driven by stiffer matrix behavior at scaffold surfaces compared to the pore interior. Results Lectin staining of decalcified scaffolds showed continued vessel growth, branching and network formation at 14 days. The fibrin gel provides no resistance to spread-out capillary networks formation, with greater vessel loops within the 450 μm pores and vessels bridging across 250 μm pores. Vessel growth in the scaffolds was observed to be stimulated by hypoxia and successive angiogenic signaling. Fibrin gels showed linear fold increase in VEGF expression and no change in BMP2. Within scaffolds, there was multiple fold increase in VEGF between days 7 and 14 and early multiple fold increases in BMP2 between days 3 and 7, relative to fibrin. There was evidence of yap/taz based hippo signaling and mechanotransduction in the scaffold groups. The vessel growth models determined by computational modeling matched the trends observed experimentally. Conclusion The differing nature of hypoxia signaling between scaffold systems and mechano-transduction sensing matrix mechanics were primarily responsible for differences in osteogenic cell and microvessel growth. The computational model implicated scaffold architecture in dictating branching morphology and strain in the hydrogel within pores in dictating vessel lengths.more » « less
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Abstract Collagen I interactions with integrins α1and α2are known to support human mesenchymal stem cell (hMSC) osteogenesis. Nonetheless, elucidating the relative impact of specific integrin interactions has proven challenging, in part due to the complexity of native collagen. In the present work, we employed two collagen‐mimetic proteins—Scl2‐2 and Scl2‐3— to compare the osteogenic effects of integrin α1versus α2signaling. Scl2‐2 and Scl2‐3 were both derived from Scl2‐1, a triple helical protein lacking known cell adhesion, cytokine binding, and matrix metalloproteinase sites. However, Scl2‐2 and Scl2‐3 were each engineered to display distinct collagen‐based cell adhesion motifs: GFPGER (binding integrins α1and α2) or GFPGEN (binding only integrin α1), respectively. hMSCs were cultured within poly(ethylene glycol) (PEG) hydrogels containing either Scl2‐2 or Scl2‐3 for 2 weeks. PEG‐Scl2‐2 gels were associated with increased hMSC osterix expression, osteopontin production, and calcium deposition relative to PEG‐Scl2‐3 gels. These data indicate that integrin α2signaling may have an increased osteogenic effect relative to integrin α1. Since p38 is activated by integrin α2but not by integrin α1, hMSCs were further cultured in PEG‐Scl2‐2 hydrogels in the presence of a p38 inhibitor. Results suggest that p38 activity may play a key role in collagen‐supported hMSC osteogenesis. This knowledge can be used toward the rational design of scaffolds which intrinsically promote hMSC osteogenesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2594–2604, 2018.
