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Direct Conversion of Human Fibroblasts to Induced Neurons

Pfisterer, Ulrich LU (2014) In Lund University, Faculty of Medicine Doctoral Dissertation Series 2014:95.
Abstract (Swedish)
Popular Abstract in Swedish

Celler som har förmågan att ge upphov till nästan alla typer av mogna celler i en organism kallas för pluripotenta stamceller. Förutom deras förmåga att bilda olika specialiserade celltyper har pluripotenta stamceller även den unika förmågan att genomgå ett obegränsat antal celldelningar.

Under utvecklingen av en organism genomgår pluripotenta stamceller en process som kallas differentiering. Under denna process erhåller varje stamcell egenskaper som är specifika för just en viss celltyp som gör att cellen kan utöva sina funktioner som till exempel en hjärtcell eller nervcell. Efter differentieringen förlorar dessa celler sin förmåga till obegränsad celldelning.

Under en lång... (More)
Popular Abstract in Swedish

Celler som har förmågan att ge upphov till nästan alla typer av mogna celler i en organism kallas för pluripotenta stamceller. Förutom deras förmåga att bilda olika specialiserade celltyper har pluripotenta stamceller även den unika förmågan att genomgå ett obegränsat antal celldelningar.

Under utvecklingen av en organism genomgår pluripotenta stamceller en process som kallas differentiering. Under denna process erhåller varje stamcell egenskaper som är specifika för just en viss celltyp som gör att cellen kan utöva sina funktioner som till exempel en hjärtcell eller nervcell. Efter differentieringen förlorar dessa celler sin förmåga till obegränsad celldelning.

Under en lång tid trodde man att differentiering av en pluripotent stamcell till en specialiserad celltyp var en irreversibel process. Dock visade upptäckten av en process som kallas nukleär reprogrammering att det är möjligt att få differentierade celler att återgå till pluripotenta stamceller. Denna upptäckt belönades med Nobelpriset i Fysiologi/Medicin 2012. Under åren har man även karakteriserat en rad faktorer som möjliggör direkt omvandling av en specialiserad cell till en annan celltyp, som till exempel tranformationen av hudceller från möss till nervceller. Detta kallas för inducerade nervceller (iNs).

I denna avhandling har jag för första gången visat en direkt omvandling av humana hudceller till humana inducerade nervceller (hiNs). Denna process kallas för direkt neuronal konvertering och leder till fullständig förändring av en hudcell till nervcell med avseende på morfologi (form och uppbyggnad) samt förmågan att ta emot och överföra nervimpulser som nervceller. Jag har även visat att dessa konverterade celler kan tas från cellodling och transplanteras i hjärnan på råttor där dessa celler överlever och formar nervceller.

Vidare har jag i denna avhandling demonstrerat att hudceller kan omprogrammeras till en specifik subtyp av nervceller så kallade dopaminerga (DA) hiNs. Detta öppnar upp möjligheten för att använda dessa celler för behandling av hjärnsjukdomar som Parkinsons sjukdom (PD). I PD dör en specifik sorts nervceller som frisätter signalsubstansen dopamin i hjärnan. Med hjälp av celltransplantation försöker man ersätta dessa nervceller med nya dopaminproducerande nervceller. Sammanfattningsvis visar denna avhandling att det är möjligt att omvandla mänskliga hudceller till dopaminproducerande nervceller som kan vara potentiella kandidater vid celltransplantationsterapier.

Denna teknik möjliggör alltså att generera ett stort antal nervceller från individers egna hudceller. Förutom att användas vid celltransplantation kan man även använda tekniken för att skapa sjukdomsmodeller. Då det är svårt att utföra biopsi (provtagning) på den humana hjärnan utan att orsaka skada, öppnar alltså tekniken upp för nya möjliga strategier inom medicinsk forskning som kan användas för sjukdomsmodellering där patientens egna hudceller programmeras om till nervceller som man sedan kan studera. På sikt kan detta leda till nya behandlingsmetoder för neurodegenerativa sjukdomar samt framställning av läkemedel som är specifika för patienten. (Less)
Abstract
During direct cellular reprogramming, forced expression of key transcription factors (TFs) directly converts one terminally differentiated cell type into that of another fate, exemplified in this theses by the conversion of fibroblasts into functional induced neurons (iNs).

Direct conversion of mouse fibroblasts to functional neurons was established in 2010, and the starting point of my doctoral thesis was the aim to transfer iN-technology to human cells and to explore the potential of this technique in regards to generate subtype-specific neurons as well as to use these cells for transplantation studies. In Paper I, we described the possibility to convert human fetal and postnatal fibroblasts into functional human induced... (More)
During direct cellular reprogramming, forced expression of key transcription factors (TFs) directly converts one terminally differentiated cell type into that of another fate, exemplified in this theses by the conversion of fibroblasts into functional induced neurons (iNs).

Direct conversion of mouse fibroblasts to functional neurons was established in 2010, and the starting point of my doctoral thesis was the aim to transfer iN-technology to human cells and to explore the potential of this technique in regards to generate subtype-specific neurons as well as to use these cells for transplantation studies. In Paper I, we described the possibility to convert human fetal and postnatal fibroblasts into functional human induced neurons (hiNs). Generating a mixed population of glutamatergic and GABAergic hiNs, we were exploring the ability to direct hiNs towards a distinct neuronal subtype and therefore selected TFs, which are expressed in midbrain dopaminergic (mDA) neurons and their progenitors. When over-expressing these fate-specifying TFs during direct neuronal conversion, we could show for the first time, that hiNs acquire a dopaminergic (DA) subtype.

This led us to explore, whether functional hiNs could be generated from adult sources. In Paper II, we succeeded in generating functional hiNs from fibroblasts of different adult donors; in addition we found that the donor age does not affect the conversion potential of the fibroblasts.

The successful generation of functional hiNs in vitro caught our interest to whether new neurons could be obtained also via direct conversion in vivo. In Paper III, we demonstrated that transplanted fibroblasts convert to hiNs in the adult rat brain and that residing astrocytes in the mouse brain can be converted into iNs.

For hiNs to become a clinically relevant cell source for cell therapy, additional analysis regarding their survival and maturation after transplantation but also the improvement of conversion efficiencies is important. In Paper IV, we addressed this in part by devising a more efficient conversion protocol as well as by examining the impact of different maturation times of hiNs in culture prior to grafting. Besides using hiNs for transplantation studies, in Paper V, we demonstrated that hiNs could be used to develop a phenotypic screening assay for the identification of small molecules and associated signaling pathways, important for direct neuronal conversion.

Overall, this thesis work has opened up possibilities to generate hiNs for brain repair, to obtain patient-specific hiNs for disease-modeling in vitro and to utilize hiNs in high content screening assays. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • PhD Wernig, Marius, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Cellular Reprogramming, Direct Conversion, Human Induced Neurons, Dopaminergic, Cell Replacement Therapy, Lentiviral Vectors
in
Lund University, Faculty of Medicine Doctoral Dissertation Series
volume
2014:95
pages
143 pages
publisher
Developmental Neurobiology
defense location
Segerfalksalen, Wallenberg Neuroscience Center, Lund
defense date
2014-09-19 13:00
ISSN
1652-8220
ISBN
978-91-7619-024-1
language
English
LU publication?
yes
id
e82adb0a-d4cc-458e-a56e-cb71c1383f86 (old id 4589297)
date added to LUP
2014-08-27 12:37:04
date last changed
2016-09-19 08:44:48
@phdthesis{e82adb0a-d4cc-458e-a56e-cb71c1383f86,
  abstract     = {During direct cellular reprogramming, forced expression of key transcription factors (TFs) directly converts one terminally differentiated cell type into that of another fate, exemplified in this theses by the conversion of fibroblasts into functional induced neurons (iNs). <br/><br>
Direct conversion of mouse fibroblasts to functional neurons was established in 2010, and the starting point of my doctoral thesis was the aim to transfer iN-technology to human cells and to explore the potential of this technique in regards to generate subtype-specific neurons as well as to use these cells for transplantation studies. In Paper I, we described the possibility to convert human fetal and postnatal fibroblasts into functional human induced neurons (hiNs). Generating a mixed population of glutamatergic and GABAergic hiNs, we were exploring the ability to direct hiNs towards a distinct neuronal subtype and therefore selected TFs, which are expressed in midbrain dopaminergic (mDA) neurons and their progenitors. When over-expressing these fate-specifying TFs during direct neuronal conversion, we could show for the first time, that hiNs acquire a dopaminergic (DA) subtype. <br/><br>
This led us to explore, whether functional hiNs could be generated from adult sources. In Paper II, we succeeded in generating functional hiNs from fibroblasts of different adult donors; in addition we found that the donor age does not affect the conversion potential of the fibroblasts. <br/><br>
The successful generation of functional hiNs in vitro caught our interest to whether new neurons could be obtained also via direct conversion in vivo. In Paper III, we demonstrated that transplanted fibroblasts convert to hiNs in the adult rat brain and that residing astrocytes in the mouse brain can be converted into iNs. <br/><br>
For hiNs to become a clinically relevant cell source for cell therapy, additional analysis regarding their survival and maturation after transplantation but also the improvement of conversion efficiencies is important. In Paper IV, we addressed this in part by devising a more efficient conversion protocol as well as by examining the impact of different maturation times of hiNs in culture prior to grafting. Besides using hiNs for transplantation studies, in Paper V, we demonstrated that hiNs could be used to develop a phenotypic screening assay for the identification of small molecules and associated signaling pathways, important for direct neuronal conversion. <br/><br>
Overall, this thesis work has opened up possibilities to generate hiNs for brain repair, to obtain patient-specific hiNs for disease-modeling in vitro and to utilize hiNs in high content screening assays.},
  author       = {Pfisterer, Ulrich},
  isbn         = {978-91-7619-024-1},
  issn         = {1652-8220},
  keyword      = {Cellular Reprogramming,Direct Conversion,Human Induced Neurons,Dopaminergic,Cell Replacement Therapy,Lentiviral Vectors},
  language     = {eng},
  pages        = {143},
  publisher    = {Developmental Neurobiology},
  school       = {Lund University},
  series       = {Lund University, Faculty of Medicine Doctoral Dissertation Series},
  title        = {Direct Conversion of Human Fibroblasts to Induced Neurons},
  volume       = {2014:95},
  year         = {2014},
}