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Generation of Dopaminergic Neurons from Human Embryonic Stem Cells
KTH, School of Biotechnology (BIO), Gene Technology.
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Since the first successful derivation of human embryonic stem cells (hESC), rapid progress has been attained in the development of strategies in differentiation of these cells into various neural lineages, with the fundamental objective of using these cells for replacement and repair of damaged neuronal circuits in the central nervous system (CNS). Of particular interest are midbrain dopaminergic (mDA) neurons, which play a central role in regulation of voluntary movement. Degeneration or loss of function of mDA neurons in the nigrostriatal pathway is associated with Parkinson disease (PD).

Stromal-Derived Inducing Activity (SDIA) is recognized as one of the most efficient methods in restricting ESC differentiation to a dopaminergic lineage, and refers to the property of mouse stromal cell lines such as PA6 or MS5 to cause ESC to differentiate to DA neurons. Although this strategy has been extensively used to generate mDA neurons from hESC, the biochemical nature of SDIA is yet unknown. 

In the present study mDA neurons were generated from the BG01V2 hESC line by SDIA. To examine whether SDIA exerts its effect directly on hESC and is responsible for early dopaminergic induction, neural progenitor cells (NPC) were enyzmatically isolated from the co-cultures and allowed to differentiate in feeder-free conditions. The isolated cells were committed to a mesencephalic neural lineage, and were capable of maintaining their phenotype and developing into postmitotic mDA neurons in feeder-free conditions. The mDA neurons showed neuronal excitability and dopamine transporter function. The in vitro proliferation and differentiation of the NPC was also investigated by a BrDU incorporation assay.

Next, the maintenance of cellular memory and capacity for proliferation of the mesencephalic NPC was assessed. The NPC could be expanded in vitro by five-fold as neurospheres for up to two weeks while retaining their DA differentiation potential, but did not retain a stable phenotype over extended periods of time. Preliminary transplantation experiments of neurospheres in striatal lesioned animals indicated, however, that these cells could survive and conserve their phenotype in vivo.

To gain additional insight into the biochemical role of SDIA in early dopaminergic induction of hESC, the separate contributions of cell surface activity and secreted factors were examined. The data revealed that the PA6 cell surface activity promoted cell survival and was mainly responsible for enhanced neurogenesis of hESC, whereas secreted factors provided DA lineage-specific instructions.

In order to identify the soluble factors responsible for the DA phenotype-inducing component of SDIA, the gene expression profile of PA6 cells was compared to that of cell lines lacking the DA-inducing property. A number of soluble factors known to be associated with CNS development that were highly expressed in PA6 cells were identified as potential DA differentiation-inducing candidates. These differentially-expressed genes included stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1 (EFNB1). When these factors, termed SPIE, were applied to the hESC, they induced dopaminergic neuronal differentiation of hESC line, BG01V2 and other karyotypically normal hESC lines in vitro. Thus, it appears that SPIE comprises the DA phenotype-inducing property of SDIA. This may provide a simple and direct means of differentiating hESC to form DA neurons in a single step, without a requirement for co-culture, animal cell lines, or animal products.

Place, publisher, year, edition, pages
Stockholm: KTH , 2008. , 83 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:25
Keyword [en]
Human, dopaminergic, embryonic stem cells, differentiation, stromal cells, PA6 cells, dopamine, neurons
National Category
Neurosciences Physiology Physiology
Identifiers
URN: urn:nbn:se:kth:diva-9591ISBN: 978-91-7415-136-7 (print)OAI: oai:DiVA.org:kth-9591DiVA: diva2:126660
Public defence
2008-12-02, FA32, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC 20100916Available from: 2008-11-20 Created: 2008-11-19 Last updated: 2010-09-16Bibliographically approved
List of papers
1. Dopaminergic neurons derived from BG01V2: a variant of human embryonic stem cell line BG01
Open this publication in new window or tab >>Dopaminergic neurons derived from BG01V2: a variant of human embryonic stem cell line BG01
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2008 (English)In: Restorative Neurology and Neuroscience, ISSN 0922-6028, Vol. 26, no 6, 447-458 p.Article in journal (Refereed) Published
Abstract [en]

 Background and Purpose: Human embryonic stem cells (hESC) areconsidered a renewable source of dopamine producing neurons, and are of particularinterest for their potential clinical use in Parkinson’s disease. In this study, wecharacterized human dopaminergic neurons generated by stromal-derived inducingactivity (SDIA) from BG01V2, a strain of human embryonic stem cell line, BG01,characterized by a chromosome 17 trisomy. Similar chromosomal changes have beenrepeatedly observed in hESC cultures in different laboratories, indicating the importanceof chromosome 17 for growth and adaptation of hESC to culture. Methods: Weinvestigated in vitro proliferation of differentiating cells using a BrDU incorporationassay, and monitored the cell population in long term cultures. Despite the cytogeneticabnormality, TH+ neurons were postmitotic at all stages of differentiation. After 30 daysof differentiation, cell division ceased in 91% of the overall population of cells in theculture, indicating intact cell cycle regulation. Results: Expression of midbrain specificmarker genes (Otx2, Pax5, Msx-1) showed differentiation of hESC-derived neuralprogenitor cells into midbrain specific dopamine neurons. These neurons expressed thedopamine transporter (DAT), and displayed functional DAT activity and electricalexcitability. Conclusions: TH+ cells derived from the BG01V2 hESC line using SDIAare postmitotic and have functional characteristics of normal dopaminergic neurons.

Keyword
SDIA, embryonic stem cells, human, dopaminergic neuron, PA6 cells
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-9612 (URN)000262162900001 ()
Note
QC 20100916. Uppdaterad från in press till published (20100916).Available from: 2008-11-20 Created: 2008-11-20 Last updated: 2010-09-16Bibliographically approved
2. Limited proliferation capacity of mesencepahlic neural progenitor cells derived from human embryonic stem cells
Open this publication in new window or tab >>Limited proliferation capacity of mesencepahlic neural progenitor cells derived from human embryonic stem cells
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(English)Manuscript (Other academic)
Abstract [en]

Midbrain neural progenitor cells (NPC) derived from human embryonic stem cells(hESC) may be useful for development of novel transplantation and gene deliverystrategies. One of the major goals of human transplantation research has been to developa means of generating dopaminergic (DA) neurons in vitro, which can be employed fortransplantation. NPC can be generated from hESC using a number of strategies. Thephenotypic conservation, stability, and differentiation of NPC generated under variousconditions are, however, not well understood. In the present study we generatedexpandable mesencephalic-restricted human NPC from the hESC line BG01V2 under theinfluence of stromal-derived inducing activity (SDIA), and assessed their capacity forproliferation and maintenance of cellular memory. The NPC could be expanded by fivefoldas neurospheres for up to 2 weeks in vitro while retaining their DA differentiationpotential, without a substantial loss of cellular memory and viability. Although cellswere continuously maintained under the influence of the midbrain patterning factors SHHand FGF8, they progressively lost their ability to differentiate to DA neurons andmaintain a stable phenotype in vitro. Preliminary transplantation experiments ofneurospheres with midbrain identity in intrastriatal 6-hydroxydopamine lesioned animalsindicated, however, that these cells could survive and conserve their phenotype in vivo.Therefore, in vitro propagation of SDIA-derived NPC under the present conditions resultsin a gradual loss of growth capacity and multipotency over time, whereas the phenotypeof transplanted NPC remains unaltered.

Keyword
Stromal-derived inducing activity, Neural progenitor cells, Neurpsphere
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-9613 (URN)
Note
QC 20100916Available from: 2008-11-20 Created: 2008-11-20 Last updated: 2010-09-16Bibliographically approved
3. Assessment of Stromal-Derived Inducing Activity in the Generation of Dopaminergic Neurons from Human Embryonic Stem Cells
Open this publication in new window or tab >>Assessment of Stromal-Derived Inducing Activity in the Generation of Dopaminergic Neurons from Human Embryonic Stem Cells
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2008 (English)In: Stem Cells, ISSN 1066-5099, Vol. 26, no 6, 1517-1525 p.Article in journal (Refereed) Published
Abstract [en]

Producing dopaminergic (DA) neurons is a major goal of human embryonic stem cell (hESC) research. DA neurons can be differentiated from hESC by coculture with the mouse PA6 stromal cell line; this differentiation-inducing effect is termed stromal-derived inducing activity (SDIA). The molecular and biochemical nature of SDIA is, however, unknown. Various studies have suggested that SDIA involves either a fixation-resistant component located on the PA6 cell surface or factors secreted into the medium by PA6 cells. To address this question, hESC were cocultured with PA6 cells for 12 days and then further differentiated with sonic hedgehog homolog, fibroblast growth factor-8, and glial cell line-derived neurotrophic factor. After 18 days, 34% of cells were tyrosine hydroxylase (TH)+. When PA6 cells were fixed or irradiated, the number of TH+ cells was decreased by threefold, whereas mitomycin-c treatment of feeder cells decreased the number of TH+ cells by 32%. The neural-inducing effect of PA6 cells, as monitored by β-III-tubulin expression, was minimally affected by mitomycin-c treatment or fixation but was decreased 50% by irradiation. Medium conditioned by PA6 cells was ineffective in differentiating TH+ cells when used alone. Conditioned medium combined with heparin and/or fixed PA6 cells produced TH+ cell differentiation, although less effectively than PA6 cell coculture. Thus, PA6 cell surface activity is required for neural differentiation of hESC, but secreted factors are required for the specific DA neuron-inducing effect.

Keyword
human embryonic stem cells, dopaminergic, PA6 cells, stromal-derived inducing activity, differentiation
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-9614 (URN)10.1634/stemcells.2008-0039 (DOI)000256907600015 ()2-s2.0-48649091029 (Scopus ID)
Note
QC 20100916Available from: 2008-11-20 Created: 2008-11-20 Last updated: 2010-09-16Bibliographically approved
4. The Molecular Nature of Stromal-Derived Inducing Activity in Dopaminergic Differentiation of Human Embryonic Stem Cells
Open this publication in new window or tab >>The Molecular Nature of Stromal-Derived Inducing Activity in Dopaminergic Differentiation of Human Embryonic Stem Cells
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(English)Manuscript (Other academic)
Abstract [en]

The utilization of Stromal-Derived Inducing Activity (SDIA) is one of the most efficientmethods in generating dopaminergic (DA) neurons from embryonic stem cells (ESC).Neuronal and dopaminergic induction can be achieved by co-culturing ESC with mousestromal cell lines, PA6 or MS5. Although it is clear that SDIA has neural inducing andmidbrain patterning activity, the molecular nature of SDIA is so far unknown. There arecontrary reports that either cell surface activity or factors secreted by PA6 cells areresponsible for SDIA. Recently, we found that PA6 cell surface and extracellular matrixmolecules primarily promoted cell survival and general neurogenesis of hESC. Incontrast, factors secreted by PA6 cells provided lineage-specific instructions in thepresence of a stabilizing factor, heparin. In the present study, in an attempt to identify thefactors responsible for dopaminergic induction of hESC, we performed cell wholegenome expression analysis to search for soluble factors produced by PA6 cells bycomparing them to various cell lines lacking the SDIA effect. Among the soluble secretedfactors differentially expressed by PA6 cells were stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1(EFNB1). The combination of these factors, which we termed SPIE, was sufficient toproduce dopaminergic neuronal differentiation of the hESC line BG01V2 and otherkaryotypically normal hESC lines in vitro.

National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-9615 (URN)
Note
QC 20100916Available from: 2008-11-20 Created: 2008-11-20 Last updated: 2010-09-16Bibliographically approved

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