generation-high-throughput-screening-biobanking-human-induced

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

University Medical Center Utrecht

To ensure the survival of engineered bone after implantation, we combined human endothelial colony forming cells (ECFCs) and multipotent stromal cells (MSCs) as a proof of concept in a co-culture model to create in vitro prevascularized bone constructs. We hypothesized that a hypoxic stimulus will contribute to prevascularization of engineered bone. Bone marrow-derived MSCs and ECFCs from human adult peripheral blood were allowed to form co-culture pellets containing ECFCs and MSCs (1:4) or MSCs only in controls. After culture under normoxia or hypoxia (5%), pellets were harvested and processed for immunohistochemistry of CD31, Î±-smooth muscle actin, and osteocalcin. Expression of vascular endothelial growth factor and SDF-1Î± was analyzed by PCR to elucidate their involvement in hypoxic stimulation of prevascularization. The normoxic condition in co-cultures of MSCs and ECFCs supported the formation and maintenance of prevascular structures, including organized CD31-positive cells embraced by differentiated mural cells. These structures failed to form in hypoxic conditions, thereby rejecting the hypothesis that hypoxia stimulates prevasculogenesis in three-dimensional engineered bone constructs. Further, the formation of prevascular structures was paralleled by increased SDF-1Î± expression. It is suggested that actual oxygen levels were below 5% in the hypoxic co-cultures, which prevented prevascular structure formation. In conclusion, our normoxic co-culture model containing cells from clinically relevant sources sustained simultaneous endothelial, smooth muscle, and osteogenic differentiation.

Hypoxia impedes vasculogenesis of in vitro engineered bone.

Pour assurer la survie d&#39;ing&eacute;nierie osseuse apr&egrave;s l&#39;implantation, nous avons combin&eacute; les cellules formant colonie endoth&eacute;liales humaines (ECFCs) et des cellules stromales multipotentes (MSCs) comme une preuve de concept dans un mod&egrave;le de co-culture de cr&eacute;er in vitro prevascularized constructions de l&#39;OS. Nous avons &eacute;mis l&#39;hypoth&egrave;se qu&#39;un stimulus hypoxique contribuera &agrave; prevascularization d&#39;os machin&eacute;. Moelle osseuse MSCs et ECFCs du sang p&eacute;riph&eacute;rique adult humain &eacute;taient autoris&eacute;s &agrave; former des boulettes de co-culture contenant ECFCs et MSCs (1:4) ou MSCs seulement chez les t&eacute;moins. Apr&egrave;s la culture sous conditions normoxiques ou hypoxie (5 %), pastilles ont &eacute;t&eacute; r&eacute;colt&eacute;es et transform&eacute;es pour immunohistochemistry CD31 actine musculaire lisse &alpha; et ost&eacute;ocalcine. Expression du facteur de croissance endoth&eacute;lial vasculaire et SDF-1&alpha; a &eacute;t&eacute; analys&eacute;e par PCR pour &eacute;lucider leur implication dans la stimulation hypoxique de prevascularization. La condition de normoxique en co-cultures de MSCs et ECFCs pris en charge la formation et le maintien des structures prevascular, y compris les cellules CD31-positive organis&eacute;es embrass&eacute;s par les cellules diff&eacute;renci&eacute;es de murales. Ces structures n&#39;a pas pu se former dans des conditions d&#39;hypoxie, rejetant ainsi l&#39;hypoth&egrave;se que l&#39;hypoxie stimule prevasculogenesis dans les constructions de l&#39;OS machin&eacute; en trois dimensions. En outre, la formation de structures prevascular a &eacute;t&eacute; accompagn&eacute;e d&#39;expression SDF-1&alpha; accrue. Il est sugg&eacute;r&eacute; que les niveaux r&eacute;els d&#39;oxyg&egrave;ne &eacute;taient inf&eacute;rieurs &agrave; 5 % dans les co-cultures hypoxiques, emp&ecirc;chant la formation des structures prevascular. En conclusion, notre mod&egrave;le de co-culture normoxique contenant les cellules cliniquement pertinente soutenue simultan&eacute; endoth&eacute;lial, le muscle lisse et la diff&eacute;renciation ost&eacute;og&eacute;nique.

Hypoxie entrave vasculog&eacute;n&egrave;se d&#39;os con&ccedil;u in vitro.

Per garantire la sopravvivenza dell&#39;osso ingegnerizzato dopo l&#39;impianto, abbiamo combinato di cellule formanti colonia endoteliali umane (ECFCs) e cellule stromali multipotenti (MSCs) come un proof of concept in un modello co-culture di creare in vitro prevascularized costrutti di osso. Abbiamo ipotizzato che uno stimolo ipossico contribuir&agrave; alla prevascularization dell&#39;osso ingegnerizzato. MSCs derivate dal midollo osseo ed ECFCs da sangue periferico umano adulto furono autorizzati a formare co-culture pellet contenenti ECFCs e MSCs (1:4) o MSCs solo nei controlli. Dopo cultura sotto fenotipi o ipossia (5%), pellet sono stati raccolti e trattati per immunoistochimica di CD31, actina muscolo liscio &alpha; e osteocalcina. Espressione del fattore di crescita vascolare endoteliale e SDF-1&alpha; &egrave; stato analizzato mediante PCR per delucidare il loro coinvolgimento nella stimolazione ipossica di prevascularization. La condizione di Normossico in co-cultures di MSCs ed ECFCs sostenuto la formazione e la manutenzione delle strutture prevascular, comprese le cellule organizzate di CD31 positivi abbracciate da cellule differenziate murale. Queste strutture non &egrave; riuscito a formare in condizioni ipossiche, respingendo cos&igrave; l&#39;ipotesi che l&#39;ipossia stimola prevasculogenesis in costrutti tridimensionali osseo ingegnerizzato. Inoltre, la formazione di strutture prevascular era parallelo da aumentata espressione SDF-1&alpha;. &Egrave; suggerito che i livelli di ossigeno effettivi erano sotto il 5% nei co-cultures ipossica, che ha impedito la formazione di struttura prevascular. In conclusione, il nostro modello di co-culture Normossico che contiene cellule provenienti da fonti clinicamente rilevanti sostenuti simultanea endoteliali, muscolari lisce e la differenziazione osteogeniche.

Ipossia ostacola vasculogenesis dell&#39;osso ingegnerizzato in vitro.

Fabrication of biomimetic tissues holds much promise for the regeneration of cells or organs that are lost or damaged due to injury or disease. To enable the generation of complex, multicellular tissues on demand, the ability to design and incorporate different materials and cell types needs to be improved. Two techniques are combined: extrusion-based bioprinting, which enables printing of cell-encapsulated hydrogels; and melt electrowriting (MEW), which enables fabrication of aligned (sub)-micrometer fibers into a single-step biofabrication process. Composite structures generated by infusion of MEW fiber structures with hydrogels have resulted in mechanically and biologically competent constructs; however, their preparation involves a two-step fabrication procedure that limits freedom of design of microfiber architectures and the use of multiple materials and cell types. How convergence of MEW and extrusion-based bioprinting allows fabrication of mechanically stable constructs with the spatial distributions of different cell types without compromising cell viability and chondrogenic differentiation of mesenchymal stromal cells is demonstrated for the first time. Moreover, this converged printing approach improves freedom of design of the MEW fibers, enabling 3D fiber deposition. This is an important step toward biofabrication of voluminous and complex hierarchical structures that can better resemble the characteristics of functional biological tissues.

Simultaneous Micropatterning of Fibrous Meshes and Bioinks for the Fabrication of Living Tissue Constructs.

Bioactive hydrogels based on naturally-derived polymers are of great interest for regenerative medicine applications. Among naturally-derived polymers, silk fibroin has been extensively explored as a biomaterial for tissue engineering due to its unique mechanical properties. Here, we demonstrate the rapid gelation of cell-laden silk fibroin hydrogels by visible light-induced crosslinking using riboflavin as a photo-initiator, in presence of an electron acceptor. The gelation kinetics were monitored by in situ photo-rheometry. Gelation was achieved in minutes and could be tuned owing to its direct proportionality to the electron acceptor concentration. The concentration of the electron acceptor did not affect the elastic modulus of the hydrogels, which could be altered by varying the polymer content. Further, the biocompatible riboflavin photo-initiator combined with sodium persulfate allowed for the encapsulation of cells within silk fibroin hydrogels. To confirm the cytocompatibility of the silk fibroin formulations, three cell types (articular cartilage-derived progenitor cells, mesenchymal stem cells and dental-pulp-derived stem cells) were encapsulated within the hydrogels, which associated with a viability >80% for all cell types. These results demonstrated that fast gelation of silk fibroin can be achieved by combining it with riboflavin and electron acceptors, which results in a hydrogel that can be used in tissue engineering and cell delivery applications.

Rapid and cytocompatible cell-laden silk hydrogel formation  riboflavin-mediated crosslinking.

Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) do not display all hallmarks of mature primary cardiomyocytes, especially the ability to use fatty acids (FA) as an energy source, containing high mitochondrial mass, presenting binucleation and increased DNA content per nuclei (polyploidism), and synchronized electrical conduction. This immaturity represents a bottleneck to their application in (1) disease modelling-as most cardiac (genetic) diseases have a middle-age onset-and (2) clinically relevant models, where integration and functional coupling are key. So far, several methods have been reported to enhance iPSC-CM maturation; however, these protocols are laborious, costly, and not easily scalable. Therefore, we developed a simple, low-cost, and rapid protocol to promote cardiomyocyte maturation using two small molecule activators of the peroxisome proliferator-activated receptor β/δ and gamma coactivator 1-alpha (PPAR/PGC-1α) pathway: asiatic acid (AA) and GW501516 (GW). METHODS AND RESULTS: Monolayers of iPSC-CMs were incubated with AA or GW every other day for ten days resulting in increased expression of FA metabolism-related genes and markers for mitochondrial activity. AA-treated iPSC-CMs responsiveness to the mitochondrial respiratory chain inhibitors increased and exhibited higher flexibility in substrate utilization. Additionally, structural maturity improved after treatment as demonstrated by an increase in mRNA expression of sarcomeric-related genes and higher nuclear polyploidy in AA-treated samples. Furthermore, treatment led to increased ion channel gene expression and protein levels.

Article	Year
Hypoxia impedes vasculogenesis of in vitro engineered bone. Tissue engineering. Part A\| PubMed ID: 21859278	2012
Simultaneous Micropatterning of Fibrous Meshes and Bioinks for the Fabrication of Living Tissue Constructs. Advanced healthcare materials\| PubMed ID: 29911317	2019
Rapid and cytocompatible cell-laden silk hydrogel formation riboflavin-mediated crosslinking. Journal of materials chemistry. B\| PubMed ID: 33001117	2020
Small molecule-mediated rapid maturation of human induced pluripotent stem cell-derived cardiomyocytes. Stem cell research & therapy\| PubMed ID: 36575473	2022

Inge Dokter

Biography

JoVE Journal Publications

Other Publications