Entwicklung von automatisierten und skalierbaren Verfahren zur Kultivierung humaner embryonaler Stammzellen

Im Rahmen dieser Dissertation wurden Verfahren zur automatisierten und zur skalierbaren Kultivierung humaner embryonaler Stammzellen (hES Zellen) erarbeitet. Hierfür wurde zunächst die manuelle humane ES Zellkultur in Gegenwart von Fibroblasten sowie unter Verwendung Fibroblasten-konditionierten Mediums etabliert. Die hierbei gewonnenen Erfahrungen bildeten die Grundlage für die Entwicklung eines automatisierten Systems für die murine und die humane ES Zellkultur, die in Kooperation mit Hamilton Life Science Robotics durchgeführt wurde. Es konnte erstmals gezeigt werden, dass ein automatisiertes System dafür geeignet ist, mES und hES Zellkulturen über einen längeren Zeitraum aufrechtzuerhalten, ohne die Proliferation oder das in vitro-Differenzierungspotenzial dieser Zellen zu beeinflussen. Die mittlere spezifische Wachstumsrate von über vier Wochen automatisiert kultivierten ES Zellen war mit 0,72 ± 0,06d-1 für mES Zellen bzw. mit 0,22 ± 0,003d-1 für hES Zellen vergleichbar zu der manuell kultivierter Zellen. 96 ± 2% der mES Zellen und 96 ± 1% der hES Zellen zeigten im Anschluss an diesen Kultivierungszeitraum eine Expression des Pluripotenz-assoziierten Markers Oct-4. Für das Oberflächenantigen SSEA-1 waren 94 ± 2% der Zellen in mES Zellkulturen immunreaktiv. hES Zellkulturen wiesen eine stabile Tra-1-60- (87 ± 4%) und Tra-1-81-Expression (89 ± 2%) auf. Der Karyotyp der automatisiert kultivierten hES Zellen blieb stabil. Des Weiteren wurde in dieser Dissertation ein skalierbarer Prozess zur Microcarrier-basierten Suspensionskultur humaner ES Zellen entwickelt. Unter Verwendung des Carriers Cytodex3 sowie eines Glasballrührers bei einer Rührerdrehzahl von 20rpm und Intervallrühren konnten mittlere spezifische Wachstumsraten von 0,34 ± 0,02d-1 erreicht werden. Diese Wachstumsrate ist mit der konventioneller Monolayerkulturen (0,26 ± 0,09d-1) mindestens vergleichbar. Über diesen Prozess konnte eine Zellpopulation gewonnen werden, die die Expression Pluripotenz-assoziierter Marker sowie ihr in vitro-Differenzierungspotenzial aufrechterhielt. Strategien zur Optimierung dieses Prozesses wurden entwickelt. Insgesamt demonstriert diese Arbeit, dass es möglich ist, humane ES Zellen bei Erhalt ihrer charakteristischen Eigenschaften unter automatisierten Bedingungen sowie über skalierbare Verfahren zu expandieren. Die in dieser Arbeit entwickelten Verfahren stellen eine Grundlage für die Entwicklung ES Zell-basierter Screeningsysteme dar. Ein skalierbarer Prozess für die standardisierte Produktion großer Zahlen an hES Zellen wäre dafür geeignet, das Ausgangsmaterial für kontrollierte Differenzierungsprozesse zur Verfügung zu stellen. Diese Differenzierungsverfahren könnten in Zukunft mit Hilfe eines automatisierten Zellkultursystems deutlich besser standardisiert werden. Die Automatisierung würde es zudem erlauben, ES Zell-abgeleitete somatische Zellen im Mikrotiterplatten-Format zu plattieren und der pharmazeutischen Industrie in validierter Form zur Verfügung zu stellen. hES Zell-abgeleitete somatische Zellpopulationen könnten es in Zukunft erlauben, die Wirksamkeit neu entwickelter Pharmaka direkt an humanen Zellen zu evaluieren, die nicht nur den Phänotyp des betroffenen Gewebes repräsentieren, sondern auch molekulare Charakteristika der betreffenden Erkrankung widerspiegeln

Cancer Genes Hypermethylated in Human Embryonic Stem Cells

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation

MicroRNA-based promotion of human neuronal differentiation and subtype specification.

MicroRNAs are key regulators of neural cell proliferation, differentiation and fate choice. Due to the limited access to human primary neural tissue, the role of microRNAs in human neuronal differentiation remains largely unknown. Here, we use a population of long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) derived from human embryonic stem cells to study the expression and function of microRNAs at early stages of human neural stem cell differentiation and neuronal lineage decision. Based on microRNA expression profiling followed by gain- and loss-of-function analyses in lt-NES cells and their neuronal progeny, we demonstrate that miR-153, miR-324-5p/3p and miR-181a/a contribute to the shift of lt-NES cells from self-renewal to neuronal differentiation. We further show that miR-125b and miR-181a specifically promote the generation of neurons of dopaminergic fate, whereas miR-181a inhibits the development of this neurotransmitter subtype. Our data demonstrate that time-controlled modulation of specific microRNA activities not only regulates human neural stem cell self-renewal and differentiation but also contributes to the development of defined neuronal subtypes

Transient modulation of microRNA activities impacts neuronal lineage development.

<p>(<b>A</b>, <b>B</b>) Histograms showing the percentage of β-III tubulin-positive cells in 7 days (<b>A</b>, ND7) and 15 days (<b>B</b>, ND15) differentiated lt-NES cells (I3 cell line) transfected with control (ctr), miR-124, miR-125b, miR-181a and miR-181a* mimics and inhibitors. (<b>C</b>) Histogram showing the percentage of TH-positive neurons in lt-NES cells transfected as described above and differentiated for 15 days. Data in <b>A</b>–<b>C</b> are presented as mean + SEM (n = 3; *, p≤0.05; **, p≤0.01; ***, p≤0.0001). (<b>D</b>) Representative immunostainings for β-III tubulin and TH in 15 days differentiated lt-NES cells transfected with ctr, miR-181a mimic or miR-181a* inhibitor. DAPI labels nuclei. Scale bar = 100 µm. (<b>E</b>) Histogram showing the percentage of TH-positive neurons in lt-NES cells differentiated for 15 days under default conditions (default) or in presence of factors that promote dopaminergic neuron differentiation (DN-factors). Data are presented as mean + SEM (n = 3; *, p≤0.05). (<b>F</b>) Histogram showing the ratio of miR-181a versus miR-181a* expression in lt-NES cells differentiated under default conditions or in presence of DN-factors, as assessed by qRT-PCR analysis. Data are normalized to RNU5A snRNA reference levels and are presented as mean + SEM (n = 3; ***, p≤0.0001). (<b>G</b>) Histogram showing the ratio of miR-181a versus miR-181a* expression in human fetal whole brain and in human fetal midbrain extracts, as assessed by qRT-PCR analysis. Data are normalized to miR-16 reference levels and presented as mean + SEM (n = 2). (<b>H</b>, <b>I</b>) Histograms showing the percentages of β-III tubulin-positive cells (<b>H</b>) and TH-positive (<b>I</b>) neurons in lt-NES cultures differentiated in presence of DN-inducing factors and transfected with miRNA mimics. The percentage of immunopositive cells in the mock-transfection controls in <b>A</b>–<b>C, H</b> and <b>I</b> is indicated by dashed lines. Abbreviations: ctr, control; DAPI, 4′,6-diamidino-2-phenylidole; DN, dopamine neuron, lt-NES, long-term self-renewing neuroepithelial-like stem cells; qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction; snRNA, small nuclear RNA; TH, tyrosine-hydroxylase.</p

Overexpression of miR-153, miR-181a/a* or miR-324-5p/3p shifts lt-NES cells from self-renewal to neuronal differentiation.

<p>(A) Quantitative real-time RT-PCR analyses showing relative expression levels of mature miR-153, miR-181a, miR-181a*, miR-324-5p and miR-324-3p in lt-NES cells (I3 cell line) transduced with either LVTHM-ctr (ctr, for endogenous levels) or the respective LVTHM-miRNA overexpressing lentiviral constructs (LVTHM-miR-153, -miR-324-5p/3p, -miR-181a/a*). Data are normalized to miR-16 reference levels and presented as mean + SEM (n = 4; *, p≤0.05; **, p≤0.01; one-tailed Student's t-test). (B) Quantification of the relative percentage of BrdU-positive cells in lt-NES cell cultures transduced with the different specified LVTHM constructs, compared to untransduced cells (indicated as “basal”, with a BrdU incorporation rate imposed to be 100%). Data are presented as mean + SEM (n = 3; *, p≤0.05). (C, D) Quantification of the percentage of β-III tubulin-positive cells in LVTHM-transduced lt-NES cell cultures after 4 days in presence of growth factors “+ GF” (C), or after 7 days in differentiation medium devoid of growth factors “− GF” (D), compared to untransduced cells (basal, dashed line). Data are presented as mean + SEM (n = 4; *, p≤0.05; **, p≤0.01). (E) Scatter plot displaying length of single neurites (in µm) for each of the conditions described above after 7 days of differentiation. Data from three independent replicates are shown. Black lines indicate average neurite length (n = 3; ***, p≤0.0001). (F, G) Immunostainings for β-III tubulin in lt-NES cells transduced with the LVTHM constructs described above, and cultured for 4 days in presence of growth factors (F, DAPI labels nuclei) or 7 days in differentiation medium devoid of growth factors (G, neurites with more than 350 µm in length are labeled in cyan-blue). All scale bars  = 100 µm. Abbreviations: BrdU, bromodeoxyuridine; ctr, control; DAPI, 4′,6-diamidino-2-phenylidole; lt-NES, long-term self-renewing neuroepithelial-like stem cells; qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction.</p

Validation of the identified microRNA expression patterns in two different cell lines.

<p>(<b>A</b>–<b>D</b>) Northern blot analyses showing expression of miRNAs in human ES cells (ES), lt-NES cells (NES) and lt-NES cells differentiated for 15 days (ND15) and 30 days (ND30) from the I3 and H9.2 cell lines. Representative miRNAs for the different expression groups identified are shown (Group 1, <b>A</b>; Group 2, <b>B</b>; Group 3, <b>C</b>). (<b>D</b>) Northern blot analyses showing expression of miR-181a and miR-181a* in the samples described above. Putative miRNA precursors are indicated by “pre”; mature miRNAs are indicated by “miR”. U6 snRNA was used as loading control. (<b>E, F</b>) qRT-PCR analyses monitoring expression of miR-153, miR-324-5p, miR-324-3p, miR-181a and miR-181a* in the samples described above from the I3 (<b>E</b>) and H9.2 (<b>F</b>) cell lines. Data are normalized to RNU5A snRNA reference levels and presented as average changes + SEM relative to expression in NES (baseline, set to 1; n = 3; *, p≤0.05; **, p≤0.005; ***, p≤0.0001). Abbreviations: ES, embryonic stem cells; lt-NES, long-term self-renewing neuroepithelial-like stem cells; qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction; snRNA, small nuclear RNA.</p

MiR-124, miR-125b and miR-181a/a*affect subspecification of lt-NES cell-derived neurons.

<p>(<b>A</b>, <b>B</b>) Immunostainings for β-III tubulin plus TH <b>(A)</b> or GAD65/67 (<b>B</b>) in lt-NES cells (I3 cell line) transduced with LVTHM-ctr or LVTHM-miR-124, -miR-125b and -miR-181a/a*, respectively, and differentiated for 15 days. Scale bars  = 100 µm. (<b>C</b>, <b>D</b>) Histograms showing the fold change in the number of TH-positive neurons (<b>C</b>) and GAD65/67-positive neurons (<b>D</b>) relative to the number of β-III tubulin-positive neurons in lt-NES cells transduced with LVTHM-ctr or LVTHM-miR-124, -miR-125b, -miR-153, -miR-181a/a* and -miR-324-5p/3p, compared to untransduced cells (equal to 1, dashed line). Data are presented as mean + SEM (n = 3; **, p≤0.01). (<b>E</b>) qRT-PCR analyses of NURR1, DAT, TH and GAD1 expression in 15 days differentiated lt-NES cell cultures overexpressing LVTHM-ctr, -miR-124, -miR-125b, and -miR-181a/a*, respectively. Data are normalized to 18S rRNA reference levels and presented as average changes + SEM relative to expression levels in LVTHM-ctr transduced lt-NES cells (equal to 1, n≥3; *, p≤0.05; **, p≤0.01; ***, p≤0.0001). Abbreviations: ctr, control; DAT, dopamine transporter; GAD, glutamic acid decarboxylase; lt-NES, long-term self-renewing neuroepithelial-like stem cells; NURR1, Nuclear receptor related 1 protein; qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction; rRNA, ribosomal RNA; TH, tyrosine-hydroxylase.</p

Analysis of microRNA expression in human ES cells, lt-NES cells and derived neural progeny.

<p>(<b>A</b>) Phase contrast image of a human ES cell colony (I3 line). (<b>B</b>–<b>D</b>) Immunofluorescent images of, respectively, self-renewing lt-NES cells stained for the neural precursor marker Nestin (<b>B</b>), and lt-NES cell cultures differentiated for 15 days (ND15; <b>C</b>), and 30 days (ND30; <b>D</b>), stained for the pan-neuronal marker β-III tubulin. DAPI labels nuclei. Scale bars = 100 µm. (<b>E</b>) Heat-map showing a hierarchical clustering of miRNA expression profiles in lt-NES cells (NES) and ND15 and ND30 differentiated neuronal cultures, compared to human ES cells (I3 ES, used as baseline). Relative expression levels in NES, ND15 and ND30 are displayed as log2 ratios compared to hES cells (yellow, expression increases; blue, expression decreases; “ns” stays for no significant expression). Representative miRNAs for the different expression groups identified are shown enlarged. In bold are indicated the newly identified neural-associated miRNAs further studied in this work. Abbreviations: DAPI, 4′,6-diamidino-2-phenylidole; ES, embryonic stem cells; lt-NES, long-term self-renewing neuroepithelial-like stem cells.</p