Generation of Artificial Blastoids Combining miR-200-Mediated Reprogramming and Mechanical Cues

Abstract

In vitro-generated blastocyst-like structures are of great importance since they recapitulate specific features or processes of early embryogenesis, thus avoiding ethical concerns as well as increasing scalability and accessibility compared to the use of natural embryos. Here, we combine cell reprogramming and mechanical stimuli to create 3D spherical aggregates that are phenotypically similar to those of natural embryos. Specifically, dermal fibroblasts are reprogrammed, exploiting the miR-200 family property to induce a high plasticity state in somatic cells. Subsequently, miR-200-reprogrammed cells are either driven towards the trophectoderm (TR) lineage using an ad hoc induction protocol or encapsulated into polytetrafluoroethylene micro-bioreactors to maintain and promote pluripotency, generating inner cell mass (ICM)-like spheroids. The obtained TR-like cells and ICM-like spheroids are then co-cultured in the same micro-bioreactor and, subsequently, transferred to microwells to encourage blastoid formation. Notably, the above protocol was applied to fibroblasts obtained from young as well as aged donors, with results that highlighted miR-200's ability to successfully reprogram young and aged cells with comparable blastoid rates, regardless of the donor's cell age. Overall, the approach here described represents a novel strategy for the creation of artificial blastoids to be used in the field of assisted reproduction technologies for the study of peri- and early post-implantation mechanisms.

Keywords: ICM-like spheroids; TR-like cells; blastoids; cellular reprogramming; miR-200 family.

Figure 1

Scheme illustrating blastoid generation from young and aged skin fibroblasts using miR-200s and mechanical cues.

Figure 2

Induction of high plasticity through miR-200-mediated reprogramming: (A) After miR-200b/c transfection, fibroblasts isolated from young and aged individuals lost their typical elongated shape (T0) and became smaller in size with granular, vacuolated cytoplasm, larger nuclei, and formed distinguishable aggregates (miR-200) (scale bars: 100 µm). (B) Transcription levels for the pluripotent-related genes OCT4, NANOG, REX1, and SOX2 in young (green bars) and aged (red bars) untreated fibroblasts (T0) and in fibroblasts transfected with miR-200 (miR-200). Gene expression is presented with the highest expression set to 1 and all others relative to this. Different superscripts indicate significant differences (p < 0.05). (C) Immunostainings show immunopositivity of miR-200-reprogrammed young and aged cells for the pluripotency-related marker OCT4. Nuclei are stained blue (scale bars: 100 μm).

Figure 3

Generation of TR-like cells from miR-200-reprogrammed cells: (A) At day 11 of trophoblast induction, young and aged cells acquired a tight adherent epithelial morphology, exhibiting a round or ellipsoid shape with round nuclei and well-defined borders (scale bars: 100 µm). (B) Transcription levels for TR-related genes (GCM1, KRT19, PGF, CYP11A1, CGA, ESRRB, CGB, HSD17B1) in untreated young (green bars) and aged (red bars) fibroblasts (T0) and at day 11 of trophoblast induction (miR200). Gene expression levels are reported, with the highest expression set to 1 and all others relative to this. Different superscripts indicate significant differences (p  <  0.05). (C) Immunostainings show young and aged cell positivity for the TR marker KRT19 (red). Nuclei are stained blue (scale bars: 100 μm).

Figure 4

Creation of ICM-like spheroids from miR-200-reprogrammed cells: (A) Young and aged fibroblasts transfected with miR-200 and encapsulated in PTFE micro-bioreactors form 3D spherical structures (scale bars: 100 μm). (B) Expression levels for the pluripotent-related genes OCT4, NANOG, REX1, and SOX2 in young (green bars) and aged (red bars) untreated fibroblasts (T0) and in fibroblasts transfected with miR-200 and encapsulated in PTFE micro-bioreactors (miR.200). Gene transcription values are reported with the highest expression set to 1 and all others relative to this. Different superscripts denote significant differences (p  <  0.05). (C) Immunostainings show immunopositivity of cells isolated from young and aged ICMs for the pluripotency-related marker OCT4. Nuclei are stained blue (scale bars: 100 μm).

Figure 5

Generation of blastoids by assembling TR-like cells and ICM-like spheroids obtained from miR-200-reprogrammed cells: (A,B) Representative images of young and aged blastoids cultured in PTFE ((A) scale bars: 100 μm) and in micro-wells ((B) scale bars: 100 μm). (C) Rates of young (green bars) and aged (red bars) blastoids showing diameters ranging from 100 to 150 μm and from 151 to 200 μm. Superscripts ^a,b indicate significant differences (p  <  0.05). (D) Representative immunostaining of young and aged blastoids showing CDX2+ cells (red) localized to the outer layer of the spheroids and OCT4+ cells (green) in the inner compartment. Nuclei are stained blue (scale bars: 100 μm). (E) Transcription levels for pluripotency- (OCT4, NANOG, REX1, SOX2) and TR-related genes (GCM1, KRT19, PGF, CYP11A1, CGA, ESRRB, CGB, HSD17B1) in TROP2+ and TROP2- young (green bars) and aged (red bars) cells. Gene transcription is indicated with the highest expression set to 1 and all others relative to this.