Human blastoid formation using hEPSCs and hPSCs and via somatic cell reprogramming. The resulting human blastoids are similar to natural blastocysts and exhibit the developmental potential to form structures resembling amniotic cavities and yolk sacs. hEPSCs: human expanded potential stem cells; hPSCs: human pluripotent stem cells; TSCs: trophoblast stem cells; EPI: epiblast; TE: trophectoderm; PrE: primitive endoderm; OKSM: Oct4, Klf4, Sox2, and c-Myc; iBlastoid: induced blastoid
.
Sozen et al. attempted to generate human blastoids using hEPSCs that were converted from human pluripotent stem cells (hPSCs)
[80][83][92]. The converted hEPSCs formed dome-shaped colonies after more than five passages using a previously established LCDM medium
[83]. After seeding the hEPSCs (four to five cells) in AggreWell 400, the hEPSCs readily formed aggregates. To facilitate aggregate formation, a medium containing Bmp4, CHIR99021 (Wnt agonist), FGF2, and Y-27632 (ROCK inhibitor) was employed. Additionally, A83-01 (ALK5 kinase inhibitor) was used to promote TE differentiation
[99]. Within the initial 48 h of 3D culture, the enrichment of F-ACTIN and PARD6 (a polarity marker gene) at the apical surface of the structures was observed, confirming the occurrence of polarization. On day 6 of culture, blastocyst-like structures comprising three lineages–EPI, PrE, and TE—were formed. Moreover, upon subjecting these blastoids to extended culture in an IVC medium
[88], they were able to form small lumens. However, the expression of certain TE markers became impaired as development progressed in vitro, and defects in aggregate formation were observed during the latter stage of the cavity formation process.
6.2. Human Blastoid Induction via the Reprogramming of Fibroblasts
In addition to using previous approaches that involve using a combination of three blastocyst-derived stem cell types and using totipotent stem cells, Liu et al. developed a novel approach to generate induced blastoids (iBlastoids) from somatic cells through a reprogramming strategy
[91]. EPI, TE, and PrE cells emerged by day 21 during the reprogramming of the fibroblasts under the fibroblast medium condition
[100]. During the reprogramming process (by day 21), intermediate cells were transferred to AggreWell plates to induce aggregate formation, and the aggregates were then cultured for 7 days in a medium that supported early feature development. On day 6, these aggregates formed 3D blastocyst-like structures (iBlastoids). The generated iBlastoids were transcriptionally similar to natural blastocysts, and the area and diameter closely resembled those of E5–7 natural blastocysts. In vitro attachment assays
[88][90] also confirmed that the iBlastoids could recapitulate the early developmental process. Over 90% of the iBlastoids attached within 24 h, exhibiting increased size, flattening, and the formation of outgrowths that resemble those observed in natural blastocysts. On day 3 of attachment, the iBlastoids displayed the polarization of Epi-like cells and the formation of a proamniotic-like cavity. This aligns with a previous report that indicated that EPI cells in human blastocysts undergo polarization and form a proamniotic-like cavity upon in vitro attachment
[88].
6.3. Human Blastoid Formation from Primed and Naïve hPSCs
ESCs exist in at least two states that exhibit transcriptional similarities to that of the distinct stages of embryonic development. Following the fertilization of the egg by a sperm, the earliest human cells, which commit to forming the embryo, undergo a transformation into naïve pluripotent hESCs. Subsequently, when the embryo implants into the uterus, the naïve pluripotent hESCs within the embryo become ‘primed’
[101]. Naïve pluripotent hESCs exhibit a developmental resemblance to the embryonic epiblast at an earlier stage compared to ‘primed’ pluripotent hESCs
[102]. While primed pluripotent hESCs exhibit restricted differentiation potential into the embryonic lineage, naïve pluripotent hESCs demonstrate a more versatile differentiation potential, encompassing both embryonic and extraembryonic (TE and PrE) lineages
[101][103][104]. Thus, Yu et al. attempted to generate human blastoids using naïve hPSCs (both hESCs and hiPSCs)
[94] that had been cultured in 5i/L/A medium
[105].
7. Gastruloids
Blastoid and embryoid models aim to recapitulate in vivo embryogenesis through the co-culture of stem cells originating from early-stage embryos. Other approaches have been pursued to simulate early embryos and gastrula in vitro. In 2014, Martinez Arias et al. generated a 3D structure called a “gastruloid” by aggregating a small number of ESCs and inhibiting several signaling pathways
[4]. Gastruloids mimic the embryonic gastrulation process, including axis organization and germ layer specification, without the presence of extraembryonic lineages. Aggregates of approximately 300 mouse ESCs were treated with Activin A, CHIR99021 (agonists of the Wnt/β-catenin signaling), and BMP (a mesoderm inducer) to initiate primitive streak (PS) formation. Following a treatment period of 48–72 h and an analysis period that spanned up to 5 days, the resulting gastruloids exhibited significant features that resembled early mouse embryos, including symmetry breaking, axial organization, the specification of germ layers, the initiation of gastrulation, and the elongation of the axis
[4].
Van den Brink et al. developed the gastruloid formation protocol to induce organogenesis and the formation of anterior neural outgrowths
[106]. By employing single-cell RNA sequencing and spatial transcriptomics techniques, they found that gastruloids exhibited gene expression patterns characteristic of axis formation and somitogenesis, closely resembling those observed in mouse embryos
[106]. Furthermore, when embedded in a Matrigel, these gastruloids formed somites along the A-P axis
[106]. A “trunk-like structure” consisting of the neural tube, gut, and somites was also generated through the embedding of four-day-old gastruloids within a Matrigel matrix
[107]. The treatment of early-stage EPI-like aggregates with the Wnt signaling inhibitor XAV could promote the formation of anterior neural tissue in gastruloids
[107].
8. Conclusions
In summary, combining the three blastocyst-derived stem cell types (ESCs, XENCs, and TSCs) or using totipotent-like stem cells alone, along with the diverse manipulation of developmental signaling molecules, results in the formation of synthetic embryos, including blastoids and embryoids. The blastoid model demonstrates the ability to mimic key aspects of pre-implantation development, such as polarization and cavitation. Through extended culture in vitro, synthetic embryos recapitulate post-implantation development both in embryonic and extraembryonic tissues. However, limitations in accurately mimicking implantation and the development of extraembryonic lineages need to be overcome to create a more comprehensive organism model. By refining culture systems, incorporating modeling mechanical environments, and developing living cell-based maternal implantation models, we can overcome these limitations, unlock deeper insights into the complexities of early development, and open a new field for research with a living, comprehensive organism model.