Stem cell models

The research of the group “Stem cell models” focuses on the development of cellular models to understand human imprinting diseases and the development of tumors of the eye, retinoblastoma and uveal melanoma. We use human cell lines and human pluripotent stem cells (induced and embryonic) to establish such models and we employ differentiation of these cells and molecular biology techniques, like genome editing and next generation sequencing, for analysis.

Angelman syndrome

Genomic imprinting is an epigenetic process regulated by DNA methylation and resulting in parent-of-origin-specific gene expression. DNA methylation is established in only one of the parental germ lines, either the maternal or the paternal one. Once established, the DNA methylation imprint is stable throughout lifetime and differentiation. The Angelman syndrome gene, UBE3A, is active on both chromosomes in most tissues, except brain. In neurons, only the maternally inherited chromosome expresses UBE3A, whereas an epigenetic mechanism silences UBE3A on the paternal copy.  If the maternal chromosome carries a genetic defect, expression of UBE3A is lost completely and the symptoms of Angelman syndrome develop.
To understand the switch from biallelic UBE3A expression in stem cells to maternal-only expression in neurons of the human brain, we reprogrammed fibroblasts from a patient with Angelman syndrome into induced pluripotent stem cells (iPSCs). We differentiated these cells, together with two other iPSC lines with different molecular causes of Angelman syndrome, into neurons. Indeed, we observed and confirmed induction of silencing of paternal UBE3A expression upon neuronal differentiation. UBE3A locates to the synapse and the nucleus. Studying if loss of UBE3A changes gene expression on transcriptome level, we could not observe substantial differences between neurons derived from healthy control iPSCs and those derived from patients with Angelman syndrome. The function of UBE3A in the nucleus is therefore still unclear.  
A new project will investigate establishemnt of DNA methylation at the regulatory region controlling imprinted expression of UBE3A and other genes in this chromosomal region. Because the process of imprint establishment naturally occurs in oocyte and because oocytes are not amenable for research experiments, we will establish a model using iPSCs and CRISPR/Cas9 genome editing.

Fig. 1: Lack of UBE3A expression in neurons of the brain causes Angelman syndrome. The Prader-Willi / Angelman syndrome locus locates to human chromosome 15 and becomes methylated in ooytes (black lollipop) but not in sperm (white lollipop). Every cell in the embryo and developing organism carries two copies of chromosome 15, one from the mother (red, methylated) and one from the father (blue, not methylated). In neurons of the brain, UBE3A is expressed from the maternal chromosome only, whereas from the paternal chromosome a long non-coding RNA, SNHG14, is expressed, which prevents UBE3A expression from the paternal chromosome. In all other body cells, SNHG14 is not expressed and both copies of UBE3A are active. In patients with Angelman syndrome the maternal copy of UBE3A carries a defect (point mutation in UBE3A, deletion of UBE3A, etc), which results in complete loss of UBE3A expression in neurons of the brain. This causes the symptoms and typical phenotype of Angelman syndrome with lack of speech, movement problems, intellectual disability and happy attitude (among others).

Retinoblastoma

In humans, biallelic inactivation of RB1 results in the development of retinoblastoma. This is a tumor of the developing neural retina occurring in children under the age of five. RB1 is also an imprinted gene, with DNA methylation on the maternal allele resulting in an expression bias in favor of the maternal allele. For an imprinted gene, we would expect parent-specific effects on tumor development. Indeed, some rare mutations in RB1 are associated with a higher risk for tumor development when inherited from the father. To enable studies on parent-specific effects in retinoblastoma, we established in vitro differentiation of human pluripotent stem cells into neural retina in 3D organoids. To mimic the RB1 genomic status in heterozygous carriers and the tumor, we introduced RB1 mutations in human embryonic stem cells by CRISPR/Cas9. Currently we analyze the effects of RB1 loss on neural retina differentiation and cell type composition in the organoids.

Fig. 2: Differentiation of human embryonic stem cells into retinal organoids. The differentiation protocol starts with human embryonic stem cells (hESCs) growing in colonies, which are suspended into a single cell solution. Seeding of a certain amount of single cells per well in multiwell plates results in the formation of a cell ball over night. By adding specific factors that influence the direction of cell differentiation, retinal layers as observed in the eye will form and self-organize into a retinal orgaonid. The bright rim visible in phase contrast at day 15 represents the retinal tissue on the outside of the organoid. By immunofluorescent staining the seven cell types of the retina can be identified. Ganglion cells form early and are present in the inner layer of the organoid, next interneurons, like horiztonal and amacrine cells, will form. Bipolar cells and Müller glia form last during differentiation. The two types of photoreceptors, cones and rods, will arise from about d90 to d152 and are located on the outer side of organoids. Differentiation of stem cells towards retinal organoids can be used to study retinal diseases, such as retinoblastoma. Figure modified from Döpper et al., Current protocols, 2020.

Uveal melanoma

Uveal melanoma is a tumor of the eye in elderly patients. Prognosis in uveal melanoma is related to chromosome 3 status: tumors with two copies have good prognosis, whereas tumors with only one copy often metastasize. Monosomy of chromosome 3 goes along with presence of mutations in the BAP1 gene, whereas tumors with disomy of chromosome 3 acquire mutations in other genes, eg EIF1AX. We started to modify these two genes in uveal melanoma cell lines using CRISPR/Cas9 and analyze the effects on DNA methylation pattern and gene expression.

Fig. 3: Uveal melanoma cell lines edited in BAP1. The BAP1 gene was targeted by CRISPR/Cas9-mediated genome editing, either in exon 1 only or in exon 1 and in exon 5. Targeting in exon 1 and exon 5 resulted in complete absence of BAP1 protein expression. A coincidental finding was the elevation of melanin production in the BAP1 negative cells, illustrated by the dark cell pellets in seven independently modfied cell lines and one control cell line.

I will move ongoing work on the described disease models to the DSMZ and develop them further in line with the scope and setting of the collection and the Human and Animal Cell Line department. I am looking forward to introduce human stem cells and 3D cellular models at the DSMZ and to develop exciting research projects with my new team.

Prof. Dr. Laura Steenpaß

Head of Department Human and Animal Cell Lines