Precise control of stem cell fate is a fundamental issue in the use of human embryonic stem (hES) cells in the context of cell therapy We examined three ways in which the microenvironment can be controlled to alter hES cell behavior, providing insight into the best conditions for maintenance of pluripotency and neural differentiation in developmental and therapeutic studies. We first examined the effects of polydimethylsiloxane (PDMS) growth surfaces on hES cell survival and maintenance of pluripotency. Lightly cured, untreated PDMS was shown to be a poor growth surface for hES cells. Some of the adverse effects caused by PDMS could be mitigated with increased curing or UV treatment of the surface, but neither modification provided a growth surface that supported pluripotent hES cells as well as polystyrene. This work provides a basis for further optimizing PDMS for hES cell culture, moving towards the use of microdevices in establishing precise control over stem cell fate. The second study explored the use of an easily constructed diffusion-based device to grow hES cells in culture on a defined, physiologic oxygen (O₂) gradient. We observed greater hES cell survival and higher levels of pluripotency markers in the lower O₂ regions of the gradient. The greatest benefit was observed at O₂ levels below 5%, narrowing the potential optimal range of O₂ for the maintenance of pluripotent hES cells. Finally, we developed a small molecule-mediated adherent and feeder-free neural differentiation protocol that reduced the cost and time scale for in vitro differentiation of neural precursors and functional neurons from human pluripotent cells. hES cell-derived neural precursors transplanted into a murine model of focal ischemic stroke survived, improved neurogenesis, and differentiated into neurons. Transplant also led to a more consistent and measurable sensory recovery after stroke as compared to untransplanted controls. This protocol represents a potentially translatable method for the generation of CNS progenitors from human pluripotent stem cells.

No description available.

Muscle aging is associated with a decrease in the number of satellite cells and their progeny, muscle progenitor cells (MPCs) that are available for muscle repair and regeneration. However, there is an increase in non-immuno-hematopoietic cells (CD45 negative) in regenerating muscle from aged mice characterized by high stem cell antigen -1(Sca-1) expression. In aged regenerating muscle, 14.2% of cells are CD45neg Sca-1pos while 7.2% of cells are CD45neg Sca-1pos in young adult muscle. In vitro, CD45neg Sca-1pos cells over express genes associated with fibrosis, potentially controlled by Wnt2. These cells are proliferative, non-myogenic and non-adipogenic, and arise in clonally-derived MPCs cultures from aged mice. Both in vitro and in vivo studies suggest that CD45neg Sca-1pos cells from aged muscle are more susceptible to apoptosis than their MPCs, which may contribute to depletion of the satellite cell pool. Therefore, with age, a subset of MPCs takes on an altered phenotype, which is marked by high Sca-1 expression. This altered phenotype prevents these cells from participating in muscle regeneration or replenishing the satellite cell pool, and instead may contribute to fibrosis in aged muscle.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 6 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2010. / Härtill 4 uppsatser.

The amniotic membrane and the amniotic fluid are one of life's most complex and delicate tissues and fluids, respectively. What was known about this tissue and fluid prior to the 20th century was extremely limited scientifically, but was significantly defined by beliefs entrenched in mysticism, folklore, and superstitions. A comprehensive literature review of the amniotic membrane tissue and amniotic fluid reveals the many unique and complex characteristics and biological properties that been heavily investigated since the turn of the 20th century and continues to surge into the 21st century. The historical perspectives, evolution, derivation, histology, structure, and composition of the amniotic membrane; and historical perspectives, volume and regulation, and cellular and non-cellular composition of the amniotic fluid are discussed here and are coalesced for an easy and comprehensible resource. Lastly, future perspectives regarding research and application of the amniotic membrane and amniotic fluid, including stem cells are discussed.

The zygote and blastomeres of cleavage stage mouse embryos have the capacity to differentiate to the embryonic and both extra-embryonic lineages and are considered functionally totipotent. Until now, it has not been possible to establish stable cell lines that resemble these totipotent-like cells. In this work, I hypothesised that by modulating signalling pathways known to be important in early embryonic development it may be possible to capture in vitro a self-renewing state that possessed features of pre-implantation blastomeres. I succeeded in formulating a novel hypothesis driven cell culture medium which allowed the establishment of a stem cell state that possessed expanded differentiation potential to the embryonic and both extra-embryonic lineages in vitro and in vivo. These cells were isolated directly from in vitro culture of mouse pre-implantation embryos or single cell blastomeres, reprogrammed from somatic cells or converted from mouse ES cells. With these cells, I generated single cell chimeras which demonstrated extensive contribution to all lineages in the developing organism providing additional evidence that this chemical medium maintained a homogenous stem cell population. I demonstrated that the transcriptome of these cells was enriched with an early pre-implantation blastomere signature, distinct from other rare published totipotent-like cells. Finally, I demonstrated that the same chemical formulation permitted the establishment in vitro of a human stem cell state that possessed expanded differentiation potential to the embryonic and extra-embryonic lineage in vitro. My work has shown for the first time that through chemical modulation of pathways implicated to be involved in pre-implantation development, a novel homogenous stem cell state that possesses a pre-implantation transcriptional signature and expanded differentiation potential to both the embryonic and extra-embryonic lineage can be established and maintained in vitro in both mouse and human, suggesting a possible interspecies conservation of the signalling networks involved in early embryonic development.

There is a huge unmet clinical need to treat damaged articular cartilage such as that caused by osteoarthritis (OA) with an estimated 8.75 million people in the UK having sought treatment for OA (ARUK 2013). Embryonic stem cells (ESCs) offer a promising alternative therapeutic approach, potentially providing an unlimited source of chondrocytes capable of regenerating the damaged cartilage however this is limited by the efficiency of the chondrogenic differentiation protocol. An improved understanding of the posttranscriptional regulation of chondrogenesis by microRNAs (miRNAs) may enable us to improve hESC chondrogenesis. Also the recent discovery that miRNAs are selectively packaged into exosomes which can then be transferred to and be functionally active within neighbouring cells suggests they may have a role in cell-cell communication. This project investigated the regulation of miRNA expression in relation to the transcriptome during hESCs-directed chondrogenesis and the possible role for exosomes during differentiation and in stem cell maintenance of hESCs. Small RNA-seq and whole transcriptome sequencing was performed on distinct stages of hESC-directed chondrogenesis using the Directed Differentiation Protocol (DDP) developed in our lab. Also small RNA-seq was performed on exosomes isolated from hESCs and chondroprogenitors along with the donor cells that the exosomes originated from. This revealed significant changes in the expression of several miRNAs during hESC-directed chondrogenesis including: upregulation of miRNAs transcribed from the four Hox complexes, known cartilage associated miRNAs and the downregulation of pluripotency associated miRNAs. Overall miRome and transcriptome analysis revealed the two hESC lines exhibited slightly different miRome and transcriptome profiles during chondrogenesis, with Man7 displaying larger changes in miRNA and mRNA expression as it progressed through the DDP suggesting it may be more predisposed to undergo chondrogenesis. Integration of miRomes and transcriptomes generated during hESC-directed chondrogenesis identified four key functionally related clusters of co-expressed miRNAs and protein coding genes: pluripotency associated cluster, primitive streak cluster, limb development cluster and an extracellular matrix cluster. Further investigation of these gene/miRNA clusters allowed the identification of several potential novel regulators of hESC-directed chondrogenesis. In accordance with the reported literature the exosomal miRNAs from hESCs and hESC-chondroprogenitors were enriched with a guanine rich motif. Notably, several of these were enriched with targets associated with embryonic skeletal system development suggesting they may play a role in regulating differentiation. Preliminary functional experiments examining pluripotency-associated exosomes suggests they may have a role in regulating hESC stem cell maintenance. However the molecular mechanism by which this is achieved has not been investigated. This research identified main miRome and transcriptome changes during hESC-directed chondrogenesis leading to the identification of several potential novel regulators of chondrogenesis and pluripotency which can be further investigated. This project has also highlighted the potential of exosomal miRNAs to regulate hESC stem cell maintenance and differentiation.

No description available.