Christian Clausen

Transient p53 Suppression Increases Reprogramming of Human Fibroblasts without Affecting Apoptosis and DNA Damage

Summary The discovery of human-induced pluripotent stem cells (iPSCs) has sparked great interest in the potential treatment of patients with their own in vitro differentiated cells. Recently, knockout of the Tumor Protein 53 (p53) gene was reported to facilitate reprogramming but unfortunately also led to genomic instability. Here, we report that transient suppression of p53 during nonintegrative reprogramming of human fibroblasts leads to a significant increase in expression of pluripotency markers and overall number of iPSC colonies, due to downstream suppression of p21, without affecting apoptosis and DNA damage. Stable iPSC lines generated with or without p53 suppression showed comparable expression of pluripotency markers and methylation patterns, displayed normal karyotypes, contained between 0 and 5 genomic copy number variations and produced functional neurons in vitro. In conclusion, transient p53 suppression increases reprogramming efficiency without affecting genomic stability, rendering the method suitable for in vitro mechanistic studies with the possibility for future clinical translation.

Patient iPSC-Derived Neurons for Disease Modeling of Frontotemporal Dementia with Mutation in CHMP2B

Summary The truncated mutant form of the charged multivesicular body protein 2B (CHMP2B) is causative for frontotemporal dementia linked to chromosome 3 (FTD3). CHMP2B is a constituent of the endosomal sorting complex required for transport (ESCRT) and, when mutated, disrupts endosome-to-lysosome trafficking and substrate degradation. To understand the underlying molecular pathology, FTD3 patient induced pluripotent stem cells (iPSCs) were differentiated into forebrain-type cortical neurons. FTD3 neurons exhibited abnormal endosomes, as previously shown in patients. Moreover, mitochondria of FTD3 neurons displayed defective cristae formation, accompanied by deficiencies in mitochondrial respiration and increased levels of reactive oxygen. In addition, we provide evidence for perturbed iron homeostasis, presenting an in vitro patient-specific model to study the effects of iron accumulation in neurodegenerative diseases. All phenotypes observed in FTD3 neurons were rescued in CRISPR/Cas9-edited isogenic controls. These findings illustrate the relevance of our patient-specific in vitro models and open up possibilities for drug target development.

Low-molecular-weight carbohydrate Pentaisomaltose may replace dimethyl sulfoxide as a safer cryoprotectant for cryopreservation of peripheral blood stem cells

Cryopreserved hematopoietic stem cell products are widely used for certain hematologic malignancies. Dimethyl sulfoxide (DMSO) is the most widely used cryoprotective agent (CPA) today, but due to indications of cellular toxicity, changes of the cellular epigenetic state, and patient‐related side effects, there is an increasing demand for DMSO‐free alternatives. We therefore investigated whether Pentaisomaltose (PIM), a low‐molecular‐weight carbohydrate (1 kDa), can be used for cryopreservation of peripheral blood stem cells, more specifically hematopoietic progenitor cell apheresis (HPC(A)) product.

Induced pluripotent stem cells (iPSCs) derived from a patient with frontotemporal dementia caused by a R406W mutation in microtubule-associated protein tau (MAPT).

Skin fibroblasts were obtained from a 59-year-old woman diagnosed with frontotemporal dementia. The disease is caused by a R406W mutation in microtubule-associated protein tau (MAPT). Induced pluripotent stem cells (iPSCs) were established by electroporation with episomal plasmids containing hOCT4, hSOX2, hKLF2, hL-MYC, hLIN-28 and shP53. iPSCs were free of genomically integrated reprogramming genes, contained the expected c.1216C>T substitution in exon 13 of the MAPT gene, expressed the expected pluripotency markers, displayed in vitro differentiation potential to the three germ layers and had normal karyotype. The iPSC line may be useful for studying hereditary frontotemporal dementia and TAU pathology in vitro.

Generation of an isogenic, gene-corrected iPSC line from a symptomatic 57-year-old female patient with frontotemporal dementia caused by a P301L mutation in the microtubule associated protein tau (MAPT) gene

Frontotemporal dementia with parkinsonism linked to chromosome 17q21.2 (FTDP-17) is an autosomal-dominant neurodegenerative disorder. Mutations in the MAPT (microtubule-associated protein tau)-gene can cause FTDP-17, but the underlying pathomechanisms of the disease are still unknown. Induced pluripotent stem cells (iPSCs) hold great promise to model FTDP-17 as such cells can be differentiated in vitro to the required cell type. Furthermore, gene-editing approaches allow generating isogenic gene-corrected controls that can be used as a very specific control. Here, we report the generation of genetically corrected iPSCs from a 57-year-old female FTD-17 patient carrying an P301L mutation in the MAPT-gene.

Generation of a human induced pluripotent stem cell line via CRISPR-Cas9 mediated integration of a site-specific heterozygous mutation in CHMP2B

Frontotemporal dementia (FTD) is an early onset neurodegenerative disease. Mutations in several genes cause familial FTD and one of them is charged multivesicular body protein 2B (CHMP2B) on chromosome 3 (FTD3), a component of the endosomal sorting complex required for transport III (ESCRT-III). We have generated an induced pluripotent stem cell (iPSC) line of a healthy individual and inserted the CHMP2B IVS5AS G-C gene mutation into both alleles, resulting in aberrant splicing. This human iPSC line provides an ideal model to study CHMP2B-dependent phenotypes of FTD3.

Induced pluripotent stem cells (iPSCs) derived from a symptomatic carrier of a S305I mutation in the microtubule-associated protein tau (MAPT)-gene causing frontotemporal dementia.

Skin fibroblasts were obtained from a 28-year-old pre-symptomatic woman carrying a R406W mutation in microtubule-associated protein tau (MAPT), known to cause frontotemporal dementia. Induced pluripotent stem cell (iPSCs) were established by electroporation with episomal plasmids containing hOCT4, hSOX2, hKLF2, hL-MYC, hLIN-28 and shP53. iPSCs were free of genomically integrated reprogramming genes, contained the expected c.1216C>T substitution in exon 13 of the MAPT gene, expressed the expected pluripotency markers, displayed in vitro differentiation potential to the three germ layers and had normal karyotype. The iPSC line may be useful for studying hereditary frontotemporal dementia and TAU pathology in vitro.

Generation of an isogenic, gene-corrected iPSC line from a pre-symptomatic 28-year-old woman with an R406W mutation in the microtubule associated protein tau (MAPT) gene

Frontotemporal dementia with parkinsonism linked to chromosome 17q21.2 (FTDP-17) is an autosomal-dominant neurodegenerative disorder. Mutations in the MAPT (microtubule-associated protein tau) gene can cause FTDP-17, but the underlying pathomechanisms of the disease are still unknown. Induced pluripotent stem cells (iPSCs) hold great promise to model FTDP-17 as such cells can be differentiated in vitro to the required cell type. Furthermore, gene-editing approaches allow generating isogenic gene-corrected controls that can be used as a very specific control. Here, we report the generation of genetically corrected iPSCs from a pre-symptomatic carrier of the R406W mutation in the MAPT-gene.

Generation of induced pluripotent stem cells derived from a 77-year-old healthy woman as control for age related diseases.

Induced pluripotent stem cells (iPSCs) hold great promise to model diseases, where the disease affected cell type is difficult to access. A major obstacle for the development of disease models is the lack of well characterized control iPSCs from old people not affected by such a disease. Furthermore, gene-editing approaches often require iPSCs from healthy donors, where pathogenic mutations can be inserted if patient material is not available. Here, we report the generation of an iPSC line (16423 #6) from a 77-year-old woman, who did not display any disease symptoms at the time, when the skin biopsy was taken.

Pentaisomaltose, an Alternative to DMSO. Engraftment of Cryopreserved Human CD34+ Cells in Immunodeficient NSG Mice:

Hematopoietic stem cell transplantation often involves the cryopreservation of stem cell products. Currently, the standard cryoprotective agent (CPA) is dimethyl sulfoxide (DMSO), which is known to cause concentration-related toxicity and side effects when administered to patients. Based on promising in vitro data from our previous study using pentaisomaltose (a 1 kDa subfraction of Dextran 1) as an alternative to DMSO for cryopreservation of hematopoietic progenitor cells (HPCs) from apheresis products, we proceeded to a preclinical model and compared the two CPAs with respect to engraftment of human hematopoietic stem and progenitor cells (HSPCs) in the immunodeficient NSG mouse model. Human HPCs from apheresis products were cryopreserved with either pentaisomaltose or DMSO, and the following outcomes were measured: (1) the post-thaw recovery of cryopreserved cells and clonogenic potential of CD34+ cells and (2) hematopoietic engraftment in NSG mice. We found that recovery and colony-forming cells data were comparable between pentaisomaltose and DMSO. The engraftment data revealed comparable human CD45+ levels in peripheral blood at 8 weeks and bone marrow at 16 weeks post transplantation. Additionally, the frequencies of CD34+CD38low/negative and myeloid/lymphoid cells in the bone marrow were comparable. We here demonstrated that long-term engrafting HSPCs were well preserved in pentaisomaltose and comparable to cells cryopreserved with DMSO. Although a clinical trial is necessary to translate these results into human use, the present data represent an important step toward the replacement of DMSO with a non-toxic alternative.