Murine neural precursor cells have been a well studied model for neural cell fate determination and stem cell function both in vivo and in primary culture. However, factors such as cell number, the presence of multiple cell populations and of niche intrinsic factors made it difficult to dissect the mechanisms regulating cortical development. To overcome this issue, we have developed a culture system where mouse embryonic stem cells (ES) are differentiated to cortical radial precursors through retinoic acid treatment of embryoid bodies. One day after plating in neural differentiation conditions, ~70% of cells in the culture are cortical radial precursors (RPs) as indicated by the definitive cortical marker Emx1, and over 8 days in culture, these RPs differentiate to pyramidal glutamatergic neurons of the cortex mimicking in vivo development. Astrocyte differentiation can be observed later as the culture progresses, which again mimics the typical timed genesis of cells in the cortex. The stem cell properties and cell fate of these RPs can be manipulated with growth factors in culture as they are in vivo. In particular, FGF2 promotes proliferation and survival, while ciliary neurotrophic factor (CNTF) induces precocious astrocyte formation. Thus, our ES-derived cortical RP cultures can serve as an alternate and complementary in vitro model to examine neural precursor biology during early development.

Murine neural precursor cells have been a well studied model for neural cell fate determination and stem cell function both in vivo and in primary culture. However, factors such as cell number, the presence of multiple cell populations and of niche intrinsic factors made it difficult to dissect the mechanisms regulating cortical development. To overcome this issue, we have developed a culture system where mouse embryonic stem cells (ES) are differentiated to cortical radial precursors through retinoic acid treatment of embryoid bodies. One day after plating in neural differentiation conditions, ~70% of cells in the culture are cortical radial precursors (RPs) as indicated by the definitive cortical marker Emx1, and over 8 days in culture, these RPs differentiate to pyramidal glutamatergic neurons of the cortex mimicking in vivo development. Astrocyte differentiation can be observed later as the culture progresses, which again mimics the typical timed genesis of cells in the cortex. The stem cell properties and cell fate of these RPs can be manipulated with growth factors in culture as they are in vivo. In particular, FGF2 promotes proliferation and survival, while ciliary neurotrophic factor (CNTF) induces precocious astrocyte formation. Thus, our ES-derived cortical RP cultures can serve as an alternate and complementary in vitro model to examine neural precursor biology during early development.

Medium spiny neurons (MSNs) play an important role in locomotion. Counterbalance between two MSN subtypes, enkephalin-positive and substance P-positive MSNs, is crucial for maintaining normal movement. Preferential degeneration of enkephalinergic MSNs in early stage Huntington’s disease (HD) contributes to abnormal involuntary movement called chorea. The reasons for this selective vulnerability are unknown. In vitro differentiation of pluripotent stem cells (PSCs) to neuronal cells is considered a potential approach for modelling neurodegenerative disorders including HD. Generation of PSC-derived enkephalinergic MSNs would be an ideal tool for dissecting their preferential degeneration. However, an enkephalinergic phenotype has never been reported in PSC-derived MSNs. We, therefore, have generated a mouse embryonic stem cell (mESC) reporter line that expresses enhanced yellow fluorescent protein (EYFP) when the cells are committed to an enkephalinergic fate. Characterisation of this mESC line via chimaera generation showed that all EYFP-positive cells were also enkephalin-positive. We have then optimised an enkephalinergic neuronal differentiation protocol using this ESC line. Interestingly, we found that a combination of Wnt inhibitor Dickkopf-related protein 1 (DKK1), sonic hedgehog (Shh) and brain-derived neurotrophic factor (BDNF), commonly used in addition to basal medium for deriving MSNs from PSCs, had a detrimental effect on enkephalin expression. Absence of these three factors, surprisingly, did not reduce the potential of ESCs to become MSNs nor did it affect the electrophysiological properties of ESC-derived MSNs. Further investigation revealed that Pre-pro-enkephalin is down-regulated in the presence of exogenous DKK1 and/or Shh but not in the presence of BDNF. We, therefore, propose that addition of exogenous DKK1 and Shh is unfavourable to derive enkephalinergic MSNs from mouse ESCs. These findings could be used to derive enkephalinergic MSNs in vitro allowing the disease in a dish approach for HD modelling.

Human pluripotent stem cells (hPSCs) have the ability to self-renew and differentiate into all specialized body cells, providing material suitable for studying basic biology, modeling disease, and for regenerative medicine. The differentiation of hPSCs into functional cell types has been further enhanced by the production of organoids, miniature 3D organ-like structures that mimic the architecture and function of their in vivo counterparts, representing more physiologically relevant models of native tissues than monolayer cultures. Our initial aim was to differentiate hPSCs into lung epithelial organoids in vitro, and we hypothesized that applying knowledge of signaling cues during embryonic development to the dish would produce lineage-specific tissue. Using a multi-stage differentiation strategy, we derived organoids sharing properties with the developing lung as well as intestine. From this work, we learned the importance of purification, selection, and using singularized precursor cells to produce populations of bona fide lineage-restricted organoids. Upon developing a type of intestinal organoid technology from hPSCs not reported before, we shifted our focus to the intestine. We generated cystic intestinal epithelial organoids called enterospheres (hEnS) in vitro from hPSCs, which mimic structural and cell type properties of the native small intestinal epithelium. hEnS growth, differentiation, and long-term culture can be controlled by modulating media conditions. Importantly, hEnS are functional in that they elicit an innate immune response upon treatment with enteric pathogens. We established hEnS as an attractive in vitro model system for studying human gastrointestinal biology. We then developed an automated hEnS imaging assay to measure responses to growth factors, bacterial products, and enteric bacteria themselves. In doing so, we demonstrated the utility of hEnS as a germ-free system for studying host-microbe interactions and intestinal maturation. Finally, we investigated the expression of protein markers of intestinal maturation in tissue sections of primary human intestine spanning gestation, and made observations that are different from those reported in mice. Overall, our work provides new and important insights into hPSC differentiation, organoid technologies, and intestinal development in humans. / Thesis / Doctor of Philosophy (PhD)

Background: Mesenchymal stromal cells (MSCs) are multipotent cells that can differentiate into various cell types and possess immunomodulatory and anti-inflammatory effects, making them interesting candidates for therapeutic applications. MSCs are present in small quantities in tissues like bone marrow and therefore need to be expanded while preserving their essential characteristics. They should adhere to plastic, differentiate into osteocytes, adipocytes and chondrocytes and express specific cell surface markers. Currently, the “golden standard” culture media supplement is fetal bovine serum (FBS). However, there is a potential contamination risk of MSCs by xenogeneic and zoonotic infectious agents, which can trigger an immune response. As an alternative, xeno-free serum supplements derived from human sources, e.g., human serum (HS) can be used.  Aim: This study aimed to identify and characterize human bone marrow derived MSCs and examine the effects different supplements have on the cells.  Methods: MSCs were cultured in 10% FBS, 2% FBS and 10% HS for 20 -21 days. Differentiation was induced and the potential was detected with immunocytochemistry. Cell surface markers CD73, CD90, CD105 and CD45 were identified with flow cytometry.  Results and Conclusion: There was no significant difference in morphology, differential potential or immunophenotype between the different serum conditions. However, HS-supplemented culture media resulted in a significantly higher number of cells with 1 x 107 cells after 20 days without affecting their differentiation potential and immunophenotype in comparison to 10% FBS with 2.2 x 106 cells (p=0.0004). MSCs cultured in 2% FBS resulted in the least number of cells (9.9 x 105) after 21 days of expansion.

The future of human embryonic stem cell (hESC) research with regards to their applicability in a therapeutic setting, relies on the development and standardisation of consistent and robust methods to demonstrate their defining characteristics; their pluripotent ability to form all three germ layers and their capacity for self-renewal. Although much research has been carried out to investigate new methods of culturing hESCs, many of these studies have not robustly concluded the impact of prolonged culture on genetic and genomic stability nor have they examined in any comparative detail the impact of the culture conditions such as differences in feeders used or the media composition in which the stem cells are cultured in. The aim of this thesis therefore was to investigate and evaluate methods for improving the uniform and robust culture and characterisation of hESCs over prolonged periods in culture. Four hESC lines ( RH5, HUES9, SHEF1 and NCL5) were chosen on the basis that they had not previously been well characterised and therefore could potentially benefit the wider stem cell community by increasing diversity, rather than continue to use the already small subset of well publicised lines. The RH5, HUES9, SHEF1 and NCL5 cells were subjected to long term passaging using recombinant enzyme TrypLE™ Express, on human feeders, mouse feeders and feeder free matrix Matrigel in combination with defined media mTeSR1, for uniform scale up. Changes in characteristic stem cell surface markers were compared using two techniques; flow cytometry and quantitative in situ fluorescence microscopy. Genomic stability was assessed by real time PCR. Chromosomal integrity was monitored using array genomic hybridisation (aCGH). Array genomic hybridisation analysis of cells cultured for 20 passages by enzymatic passaging revealed changes in copy number variations in all the stem cell lines. Aberrations on chromosomes 12, 17 and 20, appeared most commonly as a result of long term culture. Although no significant differences were seen between hESCs cultured on mouse and human feeders, cultures on Matrigel showed fewer detected chromosomal aberrations. Expression of cell surface stemness markers SSEA3, SSEA4, TRA1-60 and TRA1-81 were maintained by hESC cultured on all matrices and confirmed by the use of flow cytometry and high throughput quantitative immunofluorescence imaging using the TissueFaxs™ cell analysis microscopy system. In depth imaging revealed subtle but important differences in the way in which hESCs attach and proliferate on different matrices. Genetic profiling of each of the stem cell lines using Taqman Low density array cards to assess the expression of 96 genes by Real Time PCR, demonstrated the continued expression of stemness genes 21 at late passage, and low level expression of differentiation genes, inherent to particular stem cell lines. Although both mouse and human feeders and Matrigel support the undifferentiated growth of hESCs, subtle differences from the hESCs were seen as a result of their use, most obviously, changes in morphology and how they proliferate. This was further explored in the stem cell line NCL5, as it demonstrated a readiness to adapt to new matrices, better chromosomal stability and higher expression of cell surface markers compared with the other hESC lines. Using in vitro differentiation assays to all three germ layers, NCL5 cultured to late passage (p+20) on human feeder iMRC5, mouse feeder iMEF and feeder free matrix Matrigel, demonstrated the ability to differentiate to ectoderm, endoderm and mesoderm progenitors after induction using three 7 day flat based directed differentiation protocols. Altered differentiation patterns were detected by Real Time PCR and TissueFaxs™ imaging and quantitative analysis, as a consequence of the prolonged culture on the specific matrices used. Such key findings allude to the strong influences of microenvironment and will help to improve the standardisation of in vitro differentiation assays. From these studies, chromosomal changes had no impact on NCL5 stem cell lines‘ ability to form progenitors, however small genetic instabilities may still play a role in terminal differentiation of germ lineage specific cell types. The findings of the programme of work described has led to the successful culture methods and characterisation testing validated in this project being incorporated into routine culture and banking of research grade hESCs at the UK Stem Cell Bank. These protocols will now be made more widely available and should assist stem cell researchers in adopting the most suitable and optimum conditions for culturing stem cells in the undifferentiated and stable state. With the huge surge in stem cell research over the past decade, the development of robust characterisation and culture methods will undoubtedly have significant impact on the exploitation of these cells for regenerative medicine and to assist with this a future aim of the stem cell bank will be to standardise methodologies for clinical grade banking.

Embryonic stem (ES) cells offer a valuable source for generating insulin-producing cells. However, current differentiation protocols often result in heterogeneous cell populations of various developmental stages. Here we show the activin A-induced differentiation of mouse ES cells carrying a homologous dsRed-IRES-puromycin knock-in within the Sox17 locus into the endoderm lineage. Sox17-expressing cells were selected by fluorescence-assisted cell sorting (FACS) and characterized at the transcript and protein level. Treatment of ES cells with high concentrations of activin A for 10 days resulted in up to 19% Sox17-positive cells selected by FACS. Isolated Sox17-positive cells were characterized by defini- tive endoderm-specific Sox17/Cxcr4/Foxa2 transcripts, but lacked pluripotency-associated Oct4 mRNA and protein. The Sox17-expressing cells showed downregulation of extraembryonic endoderm (Sox7, Afp, Sdf1)-, mesoderm (Foxf1, Meox1)- and ectoderm (Pax6, NeuroD6)-specific transcripts. The presence of Hnf4α, Hes1 and Pdx1 mRNA demonstrated the expression of primitive gut/foregut cell-specific markers. Ngn3, Nkx6.1 and Nkx2.2 transcripts in Sox17-positive cells were determined as properties of pancreatic endocrine progenitors. Immunocytochemistry of activin A-induced Sox17-positive embryoid bodies revealed coexpression of Cxcr4 and Foxa2. Moreover, the histochemical demonstration of E-cadherin-, Cxcr4-, Sox9-, Hnf1β- and Ngn3-positive epithelial-like structures underlined the potential of Sox17-positive cells to further differentiate into the pancreatic lineage. By reducing the heterogeneity of the ES cell progeny, Sox17-expressing cells are a suitable model to evaluate the effects of growth and differentiation factors and of culture conditions to delineate the differentiation process for the generation of pancreatic cells in vitro. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Mammals have two orthologs, Mll and Trx2, for the Drososphila protein Trithorax (TRX), which is the founding member of the trithorax group (TrxG) of epigenetic regulators. TrxG proteins are characterized by an evolutionary conserved SET domain. A major function of all SET domain- containing proteins is to modulate gene activity, but the underlying mechanisms are poorly understood. Apparently TRX, Mll and Trx2 are histone H3 lysine 4 specific methyltransferases. So far all evidence points to roles in expression of specific target genes. However, target genes and function of the epigenetic regulator Trx2 were still unknown. Homozygous trx2 mutant embryos arrest in development because of severe and widespread defects {Glaser, 2005 #296}. Thus mouse embryonic stem (ES) cells carrying a null mutation of trx2 were used as an alternative model system to address the implication of Trx2 in differentiation. This study showed that Trx2 is redundant for ES cell self-renewal. Homozygous trx2 knockout ES cells did not exhibit cell cycle defects. However, loss of Trx2 resulted in reduced proliferation and increased apoptosis rates in trx2-/- ES cells. Due to the fact that differentiation requires an appropriate rate of population growth, trx2-/- cells were affected adversely upon in vitro differentiation. Neurogeneic differentiation of trx2 mutant cells generated fewer mature neurons than wild type cells. Moreover a temporal delay in the developmental progression to differentiation became apparent. Cardiac differentiation of trx2-/- cells confirmed the developmental defect and temporal delay. Notably differentiation of trx2-/- cells was merely delayed or impaired but it was not absent, implying that Trx2 is not required for gene expression programs specific for neurons or cardiac myocytes. We propose that differentiation of trx2-/- ES cells is impaired because apoptosis is disturbing differentiation. Apart from analyzing the phenotype of trx2 mutant cells, this work was focused on the identification of Trx2 target genes. Oligonucleotide expression arrays were used to identify genes whose expression levels were affected by the absence of Trx2. In general, loss of Trx2 function resulted in more genes with decreased than increased expression levels. This is consistent with the hypothesis that Trx2 functions as a transcriptional activator. Comparison of gene expression profiles for constitutive and conditional trx2 mutant cells enabled a distinction between direct and indirect target genes for Trx2. As a result Magoh2 was identified as the key candidate target gene for Trx2. Interaction between Trx2 and Magoh2 suggested a potential regulatory role for Trx2 in alternative splicing. Furthermore this work provided evidence that Trx2 could be involved in the maintenance of CpG island promoter gene expression, thus providing a potent regulatory mechanism for ubiquitously expressed genes.

Mesenchymal stem cells (MSCs) have demonstrated significant potential for clinical use due to their convenient isolation, lack of significant immunogenicity, lack of ethical controversy and their potential to differentiate into tissue-specific cell types. MSCs reside in almost all tissues including the adipose tissue. Adipose tissue has main advantages as wide distribution in the organism, suitable isolation and considerable amount of resident multipotent stem cells. Therefore, in this study, adipose tissue-derived mesenchymal stem cells (AT-MSCs) were isolated from BALB/c mice omentum and epididymis fat pats. During AT-MSCs maintenance and expansion in vitro, they were characterized for the expression of antigenic surface markers and for osteogenic, chondrogenic, and adipogenic differentiation potential. AT-MSCs form both sources expressed mesenchymal surface markers, CD73, and CD105 and were negative for a hematopoietic marker, CD45. The cultures derived from both adipose tissues differentiated into all three lineages. However, differences were observed in mesenchymal surface marker expression profiles as well as in the differentiation potential of AT-MSCs from different fat sources. Furthermore, AT-MSCs isolated from omentum fat depot were cultured with differentiation medium containing retinoic acid and testicular cell conditioned medium. After treatment periods, AT-MSCs showed Gdnf gene expression, this gene is a marker for Sertoli cells. The results showed that AT-MSCs from distinct fat depots have different characteristics related to stem cell surface marker expression profiles and differentiation potential. / Células-tronco mesenquimais têm demonstrado significativo potencial para aplicação terapêutica devido ao seu fácil isolamento, baixa imunogenicidade, ausência das implicações éticas e sua ampla plasticidade. Essas células estão nos mais diversos tecidos, destacando-se o tecido adiposo devido á sua ampla distribuição no organismo, conveniente obtenção e o considerável número de células-tronco mesenquimais multipotentes que podem ser isoladas desse tecido. Assim sendo, no presente estudo, células-tronco mesenquimais derivadas do tecido adiposo (AT-MSCs) foram isoladas do tecido adiposo localizado nas regiões próximas ao omento e testículos de camundongos BALB/c. Durante a manutenção e expansão das AT-MSCs in vitro, elas foram caracterizadas quanto à presença de marcadores antigênicos de superfície e potencial de diferenciação nas linhagens osteogênica, condrogênica e adipogênica. AT-MSCs de ambas as fontes expressaram os marcadores mesenquimais de superfície, CD73 e CD105, assim como foram negativas para o marcador de linhagens hematopoiéticas, CD45. Quanto ao potencial de diferenciação, os cultivos provenientes das duas origens de tecido adiposo apresentaram capacidade de diferenciar nas três linhagens acima citadas. Porém, foram observadas discretas diferenças tanto nos padrões de expressão dos marcadores mesenquimais de superfície quanto nos potenciais de diferenciação entre as AT-MSCs provenientes dos diferentes locais de deposição de gordura. Além disso, as AT-MSCs isoladas do tecido adiposo depositado em contato com o omento quando cultivadas com meios de diferenciação, contendo ácido retinóico e meio condicionado testicular demonstraram expressão do gene Gdnf o qual é reconhecidamente expresso pelas células de Sertoli. Portanto, os resultados obtidos demonstram que conforme a origem do tecido adiposo as AT-MSCs possuem diferentes características relacionadas aos marcadores de superfície assim como aos potenciais de diferenciação.

Embryonic stem (ES) cells offer a valuable source for generating insulin-producing cells. However, current differentiation protocols often result in heterogeneous cell populations of various developmental stages. Here we show the activin A-induced differentiation of mouse ES cells carrying a homologous dsRed-IRES-puromycin knock-in within the Sox17 locus into the endoderm lineage. Sox17-expressing cells were selected by fluorescence-assisted cell sorting (FACS) and characterized at the transcript and protein level. Treatment of ES cells with high concentrations of activin A for 10 days resulted in up to 19% Sox17-positive cells selected by FACS. Isolated Sox17-positive cells were characterized by defini- tive endoderm-specific Sox17/Cxcr4/Foxa2 transcripts, but lacked pluripotency-associated Oct4 mRNA and protein. The Sox17-expressing cells showed downregulation of extraembryonic endoderm (Sox7, Afp, Sdf1)-, mesoderm (Foxf1, Meox1)- and ectoderm (Pax6, NeuroD6)-specific transcripts. The presence of Hnf4α, Hes1 and Pdx1 mRNA demonstrated the expression of primitive gut/foregut cell-specific markers. Ngn3, Nkx6.1 and Nkx2.2 transcripts in Sox17-positive cells were determined as properties of pancreatic endocrine progenitors. Immunocytochemistry of activin A-induced Sox17-positive embryoid bodies revealed coexpression of Cxcr4 and Foxa2. Moreover, the histochemical demonstration of E-cadherin-, Cxcr4-, Sox9-, Hnf1β- and Ngn3-positive epithelial-like structures underlined the potential of Sox17-positive cells to further differentiate into the pancreatic lineage. By reducing the heterogeneity of the ES cell progeny, Sox17-expressing cells are a suitable model to evaluate the effects of growth and differentiation factors and of culture conditions to delineate the differentiation process for the generation of pancreatic cells in vitro. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.