# Preventing Differentiation Towards Primitive Macrophages in Stem Cells With Down Syndrome

## TL;DR

This study shows that stem cells from individuals with Down syndrome have trouble producing brain macrophage precursors, which may explain brain development issues in Down syndrome.

## Contribution

The novel finding is that trisomy 21 leads to impaired production of primitive macrophages due to resistance to mesodermal differentiation and increased apoptosis.

## Key findings

- Ts1Cje mouse embryonic stem cells and human induced pluripotent stem cells with trisomy 21 show reduced production of primitive macrophages.
- Ts1Cje-derived cells prefer endodermal differentiation and show disturbed cytokine receptor expression and increased apoptosis.
- The reduced macrophage production is transient in long-term cultures of human induced pluripotent stem cell-derived primitive macrophages.

## Abstract

Down syndrome (DS) is characterised by delayed brain development and intellectual disabilities. Brain macrophages originate from primitive macrophages that arise from mesodermal cells in the yolk sac and play a role in brain development. Previously, we showed a reduced number of brain macrophages in Ts1Cje foetuses, a murine model of DS. However, the mechanism underlying this reduction in Ts1Cje embryos remains unknown. First, in this study, we identified that the reduced brain macrophages in Ts1Cje foetuses were microglia. This study further reports the low production of primitive macrophages from Ts1Cje mouse embryonic stem cells (Ts1Cje‐mESCs). Gene expression profiling in Ts1Cje‐mESC‐derived cells indicated the persistence of pluripotency, preference for endodermal differentiation, and suppression of mesodermal differentiation. Consistently, Ts1Cje‐mESC‐derived cells showed the disturbed expression of cytokine receptors and apoptosis, and a decreased number of primitive macrophages was confirmed in the Ts1Cje yolk sac. A human induced pluripotent stem cell line established from an individual with DS also showed lower primitive macrophage production relative to diploidized controls. Thus, our data indicate resistance to mesodermal differentiation and impaired production of primitive macrophages in DS, providing valuable insights into abnormalities in brain development associated with DS.

Decreased fetal brain macrophages were previously reported in Ts1Cje mice, a model of Down syndrome (DS). We confirmed the reduction in primitive macrophages, brain macrophage precursors, using Ts1Cje‐derived mouse embryonic stem cells (Ts1Cje‐mESCs) and human induced pluripotent stem cells with trisomy 21 (Tri21‐hiPSCs), with in vivo validation. Ts1Cje‐mESCs preferred endodermal differentiation, and primitive macrophages from Ts1Cje‐mESCs expressed pro‐inflammatory‐related genes. Ts1Cje‐mESCs and Tri21‐hiPSC‐derived cells showed increased apoptosis relative to wild‐type mESCs and hiPSCs with disomy 21 (Di21‐hiPSCs). However, long‐term culture of Tri21‐hiPSC‐derived primitive macrophages revealed that this reduction was transient. These findings improve our understanding of developmental brain abnormalities in DS.