Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations.

Human pluripotent stem cells (hPS cells) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with the acquisition of large copy number variants that provide mutated cells with a growth advantage in culture. The nature, extent and functional effects of other acquired genome sequence mutations in cultured hPS cells are not known. Here we sequence the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hES cell) lines, including 26 lines prepared for potential clinical use. We then apply computational strategies for identifying mutations present in a subset of cells in each hES cell line. Although such mosaic mutations were generally rare, we identified five unrelated hES cell lines that carried six mutations in the TP53 gene that encodes the tumour suppressor P53. The TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We found that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that the P53 mutations confer selective advantage. We then mined published RNA sequencing data from 117 hPS cell lines, and observed another nine TP53 mutations, all resulting in coding changes in the DNA-binding domain of P53. In three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from the loss of heterozygosity at the TP53 locus. As the acquisition and expansion of cancer-associated mutations in hPS cells may go unnoticed during most applications, we suggest that careful genetic characterization of hPS cells and their differentiated derivatives be carried out before clinical use.NB is the Herbert Cohn Chair in Cancer Research and was partially supported by The Rosetrees Trust and The Azrieli Foundation. Costs associated with acquiring and sequencing hESC lines were supported by HHMI and the Stanley Center for Psychiatric Research. FTM, SAM, and KE were supported by grants from the NIH (HL109525, 5P01GM099117, 5K99NS08371). KE was supported by the Miller consortium of the HSCI and FTM is currently supported by funds from the Wellcome Trust, the Medical Research Council (MR/P501967/1), and the Academy of Medical Sciences (SBF001\1016)

Derivation of High Purity Neuronal Progenitors from Human Embryonic Stem Cells

The availability of human neuronal progenitors (hNPs) in high purity would greatly facilitate neuronal drug discovery and developmental studies, as well as cell replacement strategies for neurodegenerative diseases and conditions, such as spinal cord injury, stroke, Parkinson's disease, Alzheimer's disease, and Huntington's disease. Here we describe for the first time a method for producing hNPs in large quantity and high purity from human embryonic stem cells (hESCs) in feeder-free conditions, without the use of exogenous noggin, sonic hedgehog or analogs, rendering the process clinically compliant. The resulting population displays characteristic neuronal-specific markers. When allowed to spontaneously differentiate into neuronal subtypes in vitro, cholinergic, serotonergic, dopaminergic and/or noradrenergic, and medium spiny striatal neurons were observed. When transplanted into the injured spinal cord the hNPs survived, integrated into host tissue, and matured into a variety of neuronal subtypes. Our method of deriving neuronal progenitors from hESCs renders the process amenable to therapeutic and commercial use

Differentiation of hNPs into neurons after 3 weeks of maturation.

<p>A) hNP-derivates displayed a highly branched morphology, consistent with a neuronal phenotype. B) Immunolabeling with TUJ1 (green) and GFAP (red) revealed a high purity neuronal culture with few astrocytes. C) Cells were immunopositive for Tau, a microtubule associated protein, consistent with neuronal differentiation. D) Cells were immunopositive for MAP2, a microtubule associated protein, consistent with neuronal differentiation.</p

Differentiation of hNPs into neuronal subtypes after 3 weeks of maturation.

<p>A) Cells were immunopositive for ChAT, demonstrating that the majority of hNPs have the potential to become cholinergic neurons. B) 5HT expression was detected in a subset of cells, demonstrating that hNPs have the potential to become serotonergic neurons. C) GABA_A receptor α1 expression was detected in a subset of cells, demonstrating the ability to become GABA-responsive neurons. D) TH expression was detected in a subset of cells, demonstrating the ability to become dopaminergic neurons. E) DARPP32 expression was detected in a subset of cells, demonstrating the ability to become striatal interneurons. F) Few GFAP positive cells were identified in matured cultures.</p

Culture manipulation altered the percentage of neuronal subtype derivates.

<p>In RA free conditions with the addition of factors (A), DARPP32 expression was detected in a subset of cells (B), demonstrating the ability to become GABA-responsive neurons. In RA free conditions with the addition of factors (C), 5-HT expression was detected in a subset of cells (D), demonstrating the ability to become serotonergic neurons. In RA free conditions with the addition of BDNF, ChAT expression was detected in a subset of cells (E), demonstrating the ability to become cholinergic neurons. F) In RA free conditions with the addition of BDNF, 5-HT and ChAT colocalize. G) Alteration of growth factors in RA-free conditions changes ChAT expression. In RA free conditions with the addition of FGF2 (H), TH expression was detected in a subset of cells (I), demonstrating the ability to become dopaminergic neurons. J) In RA-containing conditions, few or no TH positive cells were present.</p

Differentiation of hNPs 3 months after transplantation to spinal cord injury sites.

<p>A) Human nuclei-positive cells (green) expressed TUJ1 (red), consistent with a neuronal phenotype. B) Human nuclei-positive cells (green) expressed doublecortin (red), consistent with a young neuronal phenotype. C) Human nuclei-positive cells (green) expressed p75 (red), consistent with young motor, sensory and sympathetic neurons. D) Human nuclei-positive cells (green) expressed GAD 65–67 (red), consistent with an interneuronal phenotype. E) Human nuclei-positive cells (brown) expressed ChAT (gray), consistent with a cholinergic phenotype (nuclear counterstain in purple). F) Human specific NCAM (green) and synaptophysin (red) immunolabeling suggests integration of transplanted cells with the host environment.</p

Differentiation of hESCs into neuronal progenitor cells.

<p>A) Neurospheres at day 14 of differentiation. B) After plating of neurospheres, neuroepithelial cells displayed a typical morphology by days 16–18 of differentiation. C) Musashi-1immunolabeling of cells. D) Nestin (red) and TUJ1 (green) immunolabeling of cells demonstrated a high percentage of co-localization. E) Ki67 immunolabeling revealed that a portion of hNPs are mitotic. F) Doublecortin immunolabeling revealed a high percentage of new neurons.</p

RNA expression of matured cultures is consistent with a neuronal lineage.

<p>A) Several neurogenic genes were up-regulated after 3 weeks of maturation, as compared to undifferentiated hNPs. B) S100A6 and SOX8 genes, which are often expressed in glial cells, were down-regulated in the matured cultures, as compared to undifferentiated hNPs.</p