Abstract
3D bioprinting combines cells with a supportive bioink to fabricate multiscale, multi-cellular structures that imitate native tissues. Here, we demonstrate how our novel fibrin-based bioink formulation combined with drug releasing microspheres can serve as a tool for bioprinting tissues using human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells (NPCs). Microspheres, small spherical particles that generate controlled drug release, promote hiPSC differentiation into dopaminergic neurons when used to deliver small molecules like guggulsterone. We used the microfluidics based RX1 bioprinter to generate domes with a 1 cm diameter consisting of our novel fibrin-based bioink containing guggulsterone microspheres and hiPSC-derived NPCs. The resulting tissues exhibited over 90% cellular viability 1 day post printing that then increased to 95% 7 days post printing. The bioprinted tissues expressed the early neuronal marker, TUJ1 and the early midbrain marker, Forkhead Box A2 (FOXA2) after 15 days of culture. These bioprinted neural tissues expressed TUJ1 (15 ± 1.3%), the dopamine marker, tyrosine hydroxylase (TH) (8 ± 1%) and other glial markers such as glial fibrillary acidic protein (GFAP) (15 ± 4%) and oligodendrocyte progenitor marker (O4) (4 ± 1%) after 30 days. Also, quantitative polymerase chain reaction (qPCR) analysis showed these bioprinted tissues expressed TUJ1, NURR1 (gene expressed in midbrain dopaminergic neurons), LMX1B, TH, and PAX6 after 30 days. In conclusion, we have demonstrated that using a microsphere-laden bioink to bioprint hiPSC-derived NPCs can promote the differentiation of neural tissue.
Highlights
Human induced pluripotent stem cells were discovered in 2007 when scientists determined that overexpression of certain transcription factors could revert adult human fibroblasts back into stem cells possessing the property of pluripotency (Takahashi et al, 2007)
Three different types of dome shaped bioprinted constructs containing healthy human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells (NPCs) were printed from the corresponding computer aided design (CAD) file: NPCs only treated with guggulsterone in the media as a positive control referred to as soluble guggulsterone (SG), NPCs along with blank microspheres as a negative control referred to as UM, and NPCs along with guggulsterone releasing microspheres referred to as GM
ICC was performed on constructs for all three groups for the cellular markers TUJ1 and Forkhead Box A2 (FOXA2) at day 15 (Figure 4) and on day 30 for TUJ1 and tyrosine hydroxylase (TH) (Figures 5, 6)
Summary
Human induced pluripotent stem cells (hiPSCs) were discovered in 2007 when scientists determined that overexpression of certain transcription factors could revert adult human fibroblasts back into stem cells possessing the property of pluripotency (Takahashi et al, 2007). More recent work from the McAlpine group demonstrated that multiple neural cell types, including neural stem cells derived from hiPSCs, could be printed with relatively high levels of viability into structures that resemble the spinal cord (Joung et al, 2018). Most of these bioprinting studies have not attempted to generate structures that resemble the brain
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