Table 3:

Advantages and disadvantages of T21 cellular models, relevant phenotypic findings, and responses to treatment

Cell type	Advantages	Disadvantages	Findings in Cells from Individuals with Down Syndrome	Effects of Therapeutic Molecules	References
Amniocytes & villi	Obtained from amniocentesis or chorionic villus sampling during the first & second trimester Express stem cell markers Considered maternal tissue, no major ethical issues concerning the fetus Can be grown & expanded in vitro May carry signatures that give insights of disease state	Heterogenous, mixed population of cell types More limited access & requires IRB protocols & maternal consent For amniocytes, gene expression profile changes with fetal age, which may impact experimental outcome Limited growth capacity which limits use in high throughput screening	Genome–wide gene expression dysregulation Decreased cell proliferation High prevalence of cell senescence Shorter telomeres in amniocytes Increased proteolytic activity in amniocytes Reduced gene expression of mitochondrial ATPase 6 & TFAM in amniocytes Hypertrophic & hypovascular villi	Apigenin treatment reduces oxidative stress, improves anti–oxidant capacity, induces the expression of genes that are important for G2/M transition & decreases the expression of pro–inflammatory markers	[46, 53-55]
Fibroblasts	Obtained from humans with DS throughout lifespan (fetal, infants, children, adolescents & adults) Available in cell biobanks Retain genetic defects & systemic phenotypes Can be grown & expanded in vitro Can be used for reprogramming to generate iPSCs Carry signatures that give insights of disease state	Not CNS cells; not representative of diseased tissue or CNS dysfunction Limited growth capacity reduces usefulness in high throughput drug screening. After certain number of passages, cells enter senescence. Properties change with extended time in culture	Genome–wide gene expression dysregulation Decreased cell proliferation Increased senescence Shorter telomeres Increased ROS production Reduced mitochondrial membrane potential, impaired ATP production, irregular shapes & increased mitochondrial mass. Mitochondrial dysfunction & oxidative stress exacerbated in T21 fibroblasts from individuals with heart defects Disrupted proteostasis network: increased endoplasmic reticulum (ER) stress, protein ubiquitination, & disrupted proteasome activity Increased exosome secretion via increased expression of CD63 to mitigate endosomal abnormalities Overactivation of interferon signaling response through JAK/STAT signaling Defective repair of ROS–induced DNA damage, activation of DNA damage response, & increased p53 protein expression	EGCG increases mitochondria biogenesis & rescues mitochondrial complex I, improved ATP synthase activity & suppressed oxidative stress Metformin activates PGC1α, promotes mitochondrial biogenesis, mitochondrial activity & ATP production JAK inhibitor, ruxolitinib, induced phosphorylation of STAT1, promotes cell proliferation	[56-60, 130, 131]
CNS-derived Primary Cells	Obtained from post-mortem tissues Can be grown in vitro Reflect CNS phenotypes Carry signatures that give insights of disease state	Limited growth capacity Limited access to tissues Variable and/or poor quality of post-mortem tissues	Genome-wide gene expression dysregulation Increased oxidative stress and apoptosis Increased ROS production Mitochondrial dysfunction, abnormal mitochondrial morphology, and networks	Antioxidant treatment improved viability of T21 neurons	[61, 62, 64, 65]
Lymphoblastoids:	Arise from B–lymphocytes transformed with Epstein Barr virus (EBV) Can be expanded for many months (unlike lymphocytes) without genetic rearrangements common in other immortalized cell lines Have been used in expression studies of whole genome dysregulations in T21 Reliable, inexpensive & easily accessible Can be used to detect biologically plausible correlations between candidate genes & various genetically–induced diseases May carry signatures that give insights of disease state	Lymphoid phenotype makes them inconvenient for CNS research Cell are immortalized using EBV virus	Genome–wide gene expression dysregulation Impaired proliferative capacity & increased sensitivity to genotoxic stress Mitochondrial dysfunction reduced mitochondrial membrane potential, reduced ATP production & increased ROS production Disrupted proteostasis network, reduced protein ubiquitination & increased proteasome activity Impaired autophagy enlarged early endosomes & enhanced exosome secretion Overactivation of interferon signaling	Have not been evaluated	[59, 68, 132]
iPSC, NSC, & iPSC–derived cells	Obtained by reprogramming of somatic cells using Yamanaka factors. Somatic cells are easy to obtain from patients or cell biobanks. Can be grown & expanded in vitro indefinitely making them ideal material for high throughput drug screening Can be used to generate the three embryonic layers lineages (ectoderm, mesoderm & endoderm) to study developmental events Even after reprogramming, cells retain genetic defects/chromosomal abnormalities Neural stem cells generated from iPSCs are multipotent & give rise to CNS cell types May carry signatures that give insights of disease state.	Require reprogramming using integrative or non–integrative methods Transformation efficiency varies although it has been improved nonintegrating methods High maintenance, time consuming, expensive Cultures may be heterogeneous Genetic instability reported with increased passaging requiring regular karyotyping Retain immature, fetal phenotypes Do not recapitulate complex neuronal circuits as in in vivo animal models	Global transcriptome dysregulation Increased oxidative stress Mitochondrial dysfunction Gliogenic shift with spontaneous differentiation (more astrocytes than neurons) in T21 cells Altered astrocyte–neuron communication. Hyperactivation of Akt/mTOR signaling is thought to be the trigger of this phenotype T21 iPSCs–derived astrocytes appear to be in a reactive state with more branching, thicker branches. Decreased neurite length in T21 iPSC derived neurons Altered electrophysiological properties of iPSC derived neurons and astrocytes; alterations Ca2+ signaling & decreased spontaneous post synaptic currents Decreased migration of GABAergic interneurons derived from T21 iPSCs Increased Aβ peptide generation in T21–iPSC cortical neurons	Treatment with EGCG (DYRK1A inhibitor) or DYRK1A shRNA promotes neurogenesis in T21 iPSCs derived neural progenitors & improved neuronal maturation Treatment with minocycline rescued the neuronal/gliogenic ratio, improved neurogenesis, suppressed apoptosis, & rescued neuronal electrophysiological properties in T21 derived neurons,	[77, 79, 133-138]

IRB: Institutional Review Board, CNS: central nervous system, TFAM: Transcription Factor A, mitochondrial, ROS; reactive oxygen species, PGC1α: Peroxisome proliferator-activated receptor-γ coactivator