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Stem cells, gene editing and organoid platforms

Induced pluripotent stem cells (iPSCs)

The recent discovery of reprogramming human somatic cells into induced pluripotent stem cells (iPSCs) offers an innovative approach to the study of many human genetic diseases. Like human embryonic stem cells, iPS cells self-renew indefinitely and have unlimited developmental potential. The creation of patient-specific iPSCs holds great promise for understanding genetic disease mechanisms and for in vitro testing of potential therapies. See the page from EuroStemCell for more detailed information.

Overview: Via iPSC-Technology, patient specific Fibroblasts can be reprogrammed in stem cells. These cells can be used for in vitro disease modelling, drug screening campaigns and cell replacement experiments.

Utilized technologies in the facility

Fibroblasts are reprogrammed with standard viral (retroviral/lentiviral) vectors. We can work with frozen or live cultures. In the future we aim at replacing the retroviral/lentiviral system by non-integrative Sendai viruses. We work with mouse and human cells.

Procedure

  1. Fibroblast culture
  2. Mycoplasma testing
  3. Viral reprogramming
  4. Colony picking
  5. Colony expansion
  6. Quality testing
  7. Expansion or freezing

 Quality tests involve

  • ŸMycoplasma testing
  • AP stainings
  • Immunofluorescence stainings
  • PCR validation (exogenous and endogenous factors)
  • Chromosome set analysis
  • Embryonic body formation

Induced pluripotent stem cells: (A) Human induced pluripotent stem cell colony cultured under feeder free conditions. (B) The cells express iPSCs markers like Nanog (green) and SSEA4 (red), or (C) Oct4 (green) and SSEA4 (red). (D) IPSCs colonies are positive for the marker alkaline phosphatase and (E) Their chromosome number is analyzed. (F) IPSCs undergoing differentiation into embryonic bodies.

Differentation of iPSCs

Focus on neural lineage

Several differentiation approaches, starting from iPSCs are possible:

  • Undirected differentiation into embryonic bodies (all three germ layers).
  • Differentiation into neurons (mix of neuronal subtypes)
  • Specific differentiation in midbrain dopaminergic neurons.
  • Specific differentiation into astrocytes

Differentiation: (A) IPSCs can be differentiated into neurons. (B) They express the neuronal marker MAP2 (green). (C) Synaptic contacts are established (Synaptophysin - red). (D) Efficient differentiation into dopaminergic neurons is possible (Tyrosine Hydroxylase, TH – green; TuJ1 – red). (E) Differentiation into homogenous cultures of astrocytes is possible (GFAP - red).

Cell banking: IPSCs generated in our facility can be stored at the Integrated BioBank of Luxembourg.

Genetic engineering in human induced pluripotent stem cells for in vitro disease modeling

Personalized PD patient specific iPSCs have the advantage of carrying the disease inducing mutations. However, they have the drawback that aside from the PD-associated mutation the genetic backgrounds of the individuals are extremely diverse. Therefore, it is not easy to compare phenotypes observed with cells from different patients. In order to overcome the limitations caused by the variable genetic background we additionally make use of isogenic iPSCs carrying defined PD specific mutations. The comparison of personalized cells with isogenic cells will allows us to deduce phenotypes and mechanisms that are disease specific and specific for a certain mutation. Additionally, this approach probably gives us the opportunity to define unifying themes that are common to PD.

 

Technically these mutations will be introduced in human iPSC by utilization of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system. In this approach RNA with a certain secondary structure and a sequence-stretch complementary to the genomic part where a mutation should be introduce, is used. The secondary structure recruits the Cas9 endonuclease to this region and Cas9 cuts the selected genomic locus. This double-strand break is repaired by the cell using either homologous recombination or non-homologous end-joining. By simultaneously presenting plasmids as templates that contain the sequence with the desired mutation, homologous recombination will results in a genomic sequence with the desired mutation.

Publication: FACS-Assisted CRISPR-Cas9 Genome Editing Facilitates Parkinson's Disease Modeling Arias, Jonathan; Jarazo, Javier; Qing, Xiaobing; Walter, Jonas; Gomez Giro, Gemma; Nickels, Sarah Louise; Zaehres, Holm; Schoeler, Hans Robert; Schwamborn, Jens Christian in Stem Cell Reports (2017)

Patent : WO2017129811A1 - Means and methods for selecting transformed cells

 

Brainoids as model for Parkinsons disease

Cell cultures and animal experiments are proven methods in medical research. In brain research, for example, such methods reveal important insights into the causes and possible treatments of neurodegenerative diseases like Parkinson’s disease. However, not all structures and functions of the human brain can be represented with sufficient accuracy in these models.  

The model is a miniature artificial tissue culture derived from human stem cells. Skin cells donated by patients are transformed back into stem cells and these stem cells can be induced to develop into brain cells. In another, more elaborate method, they grow into three-dimensional tissue cultures with a brain-like structure. The cells follow an intrinsic programme that essentially corresponds to the early stage of human development. Over a span of several weeks, different cell types develop that are characteristic of the human midbrain; in addition to glial and auxiliary cells, there are also so-called dopaminergic neurons. The latter are of particular interest in Parkinson’s disease research. Dopaminergic neurons produce the neurotransmitter dopamine which is required for maintaining controlled body movements. In Parkinson’s disease, the dopaminergic neurons die off, and this results in tremors and other motor symptoms in patients. The 3D cell cultures, however, are valuable for more than just the presence of this specific cell type. In these so-called organoids, the cells connect together in a network, whereupon they can transmit and process signals. Due to their spatial organization, they exhibit characteristics that cannot be observed in classical flat cell cultures. The tissue cultures are of human origin. We believe that the experimental data obtained from organoids is more comparable to what’s happening in the patient than the data obtained from rat or mouse experiments. The organoids furthermore offer new approaches in research because they can be grown from skin cells taken from Parkinson’s patients and from healthy people. With these new cell cultures methods, we can study the mechanisms that lead to Parkinson’s disease much better than we could before. 

The study was funded by the Luxembourg National Research Fund (FNR) through its programmes CORE and AFR, by the University of Luxembourg, by JPND Research, and by the European Union in the scope of the H2020 project SysMedPD. 

Publication:Derivation of Human Midbrain-Specific Organoids from Neuroepithelial Stem Cells Monzel, Anna Sophia; Smits, Lisa; Hemmer, Kathrin; Hachi, Siham; Lucumi Moreno, Edinson; Van Wüllen, Thea Maria; Jarazo, Javier; Walter, Jonas; Werthschulte, Inga; Boussaad, Ibrahim; Berger, Emanuel; Fleming, Ronan MT; Bolognin, Silvia; Schwamborn, Jens Christian in Stem Cell Reports (2017)