CMGH Cellular and Molecular Gastroenterology and Hepatology, 2015
SUMMARY We show that enteric neural cells isolated from Hirsch-sprung disease patients can coloni... more SUMMARY We show that enteric neural cells isolated from Hirsch-sprung disease patients can colonize aneuronal colon tissue to generate neurons and glia. Our findings establish the therapeutic potential of using patient's own neural cells to form an enteric nervous system in autologous tissue. BACKGROUND & AIMS: Hirschsprung disease (HSCR) is caused by failure of cells derived from the neural crest (NC) to colonize the distal bowel in early embryogenesis, resulting in absence of the enteric nervous system (ENS) and failure of intestinal transit postnatally. Treatment is by distal bowel resection, but neural cell replacement may be an alternative. We tested whether aneuronal (aganglionic) colon tissue from patients may be colonized by autologous ENS-derived cells.
The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor li... more The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the GSK3β and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named 'caudal progenitor cells' (CNPs). CNPs co-express caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog or FGF2 signalling, efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally-specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from hESC include FGF8, canonical WNT and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube.
The great potential of induced pluripotent cells (iPS) cells is that it allows the possibility of... more The great potential of induced pluripotent cells (iPS) cells is that it allows the possibility of deriving pluripotent stem cells from any human patient. Generation of patient-derived stem cells serves as a great source for developing cell replacement therapies and also for creating human cellular model systems of specific diseases or disorders. This is only of benefit if there are well-established differentiation assay systems to generate the cell types of interest. This chapter describes robust and well-characterized protocols for differentiating iPS cells to neural progenitors, neurons, glia and neural crest cells. These established assays can be applied to iPS cell lines derived from patients with neurodegenerative disorders to study cellular mechanisms associated with neurodegeneration as well as investigating the regenerative potential of patient derived stem cells.
Stem cells are specialized cells that possess a capacity to undergo self-renewal while at the sam... more Stem cells are specialized cells that possess a capacity to undergo self-renewal while at the same time having the ability to give rise to at least one or more differentiated or mature cell type. They therefore represent a fundamental cornerstone during the life of all vertebrates, playing central roles in the production of new and replacement cells for tissues during development and homeostasis, including repair following disease or injury. This unit is a review of stem cells, their roles in development, and their potentials as therapeutic agents.
With the discovery two decades ago that the adult brain contains neural stem cells (NSCs) capable... more With the discovery two decades ago that the adult brain contains neural stem cells (NSCs) capable of producing new neurons, a great deal of research has been undertaken to manipulate these cells to repair the damaged nervous system. Much progress has been made in understanding what regulates adult neural stem cell specification, proliferation and differentiation but much remains to be determined. Lessons can be learned from understanding how embryonic neural stem cells produce the exquisitely complicated organ that is the adult mammalian nervous system. This review will highlight the role of transcriptional regulation of mammalian neural stem cells during embryonic development and compare these to the adult neural stem cell/neural precursor cell (NPC) niches of the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampal dentate gyrus. Normal physiological NSC/NPC regulation will be explored, as well as their regulation and responses following neural injury and disease. Finally, transcriptional regulation of the endogenous NSC/NPCs will be compared and contrasted with embryonic stem/induced pluripotent stem (ES/iPS) cell-derived NSC/NPCs. Recapitulation of the embryonic sequence of transcriptional events in neural stem cell development into specific neuronal or glial lineages improves directed differentiation of ES/iPS cells and may be useful for activation and specification of endogenous adult neural stem cells for therapeutic purposes.
Neural differentiation from embryonic stem cells involves progressive stages of neural induction,... more Neural differentiation from embryonic stem cells involves progressive stages of neural induction, expansion and maintenance of neural stem/progenitor cells, and differentiation to neurons and glia. Our understanding of the signals involved in each of these processes is primarily based on our knowledge of neural development during embryogenesis. This review will focus on the signalling pathways that have been identified to play a role in neural differentiation of human embryonic stem cells (hESCs), including their induction to neuroectoderm, maintenance and expansion of hESC-derived neurospheres, differentiation to neurons and specification to specific neuronal lineages. Understanding the signals involved in each of these stages is important for optimising methods to derive specific cell types for transplantation therapies, as well as for providing insight into the mechanisms of human neurogenesis.
The reported pluripotential capabilities of many human stem cell types has made them an attractiv... more The reported pluripotential capabilities of many human stem cell types has made them an attractive area of research, given the belief they may hold considerable therapeutic potential for treating a wide range of human diseases and injuries. Although the bulk of stem cell based research has focused on developing procedures for the treatment of pancreatic, neural, cardiovascular and haematopoietic diseases, the potential for deriving respiratory cell types from stem cells for treatment of respiratory specific diseases has also been explored. It is suggested that stem cell derivatives may be used for lung replacement/regeneration therapeutics and high though-put pharmacological screening strategies for a variety of respiratory injuries and diseases including: cystic fibrosis, chronic obstructive pulmonary disease, respiratory distress syndrome, pulmonary fibrosis and pulmonary edema. This review will explore recent progress in characterizing adult respiratory and bone marrow derived stem cells with respiratory potential as well as the endogenous mechanisms directing the homing of these cells to the diseased and injured lung. In addition, the potential for embryonic stem cell based therapies in this domain as well as the histological, anatomical and molecular aspects of respiratory development will be summarized.
Human embryonic stemlike cells (hESCs) are pluripotent cells derived from blastocysts. Differenti... more Human embryonic stemlike cells (hESCs) are pluripotent cells derived from blastocysts. Differentiating hESCs into respiratory lineages may benefit respiratory therapeutic programs. We previously demonstrated that 24% of all mouse embryonic stem cell (mESC) derivatives cocultured with embryonic day 11.5 (E11.5) mouse lung rudiments display immunoreactivity to the pneumonocyte II specific marker surfactant-associated protein C (Sftpc). Here we further investigate the effects of this inductive niche in terms of its competence to induce hESC derivative SFTPC immunoreactivity and the expression of other markers of terminal lung secretory units. When hESCs were cocultured as single cells, clumps of approximately 10 cells or embryoid bodies (EBs), hESC derivatives formed pan-keratin-positive epithelial tubules at high frequency (>30% of all hESC derivatives). However, human-specific SFTPC immunoreactivity associated with tubule formation only at low frequency (<0.1% of all hESC derivatives). Human-specific SFTPD and secretoglobin family 1A member 1 (SCGB1A1, also known as CC10) transcripts were detected by PCR after prolonged culture. Expression of other terminal lung secretory unit markers (TITF1, SFTPA, and SFTPB) was not detected at any time point analyzed. On the other hand, hESC derivatives cultured as plated EBs in media previously demonstrated to induce Sftpc expression in isolated mouse fetal tracheal epithelium expressed all terminal lung secretory unit markers examined. mESCs and hESCs thus display fundamental differences in their response to the E11.5 mouse lung inductive niche, and these data provide an important step in the delineation of signaling mechanisms capable of efficiently inducing hESC differentiation into terminal secretory units of the lung.
CMGH Cellular and Molecular Gastroenterology and Hepatology, 2015
SUMMARY We show that enteric neural cells isolated from Hirsch-sprung disease patients can coloni... more SUMMARY We show that enteric neural cells isolated from Hirsch-sprung disease patients can colonize aneuronal colon tissue to generate neurons and glia. Our findings establish the therapeutic potential of using patient's own neural cells to form an enteric nervous system in autologous tissue. BACKGROUND & AIMS: Hirschsprung disease (HSCR) is caused by failure of cells derived from the neural crest (NC) to colonize the distal bowel in early embryogenesis, resulting in absence of the enteric nervous system (ENS) and failure of intestinal transit postnatally. Treatment is by distal bowel resection, but neural cell replacement may be an alternative. We tested whether aneuronal (aganglionic) colon tissue from patients may be colonized by autologous ENS-derived cells.
The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor li... more The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the GSK3β and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named 'caudal progenitor cells' (CNPs). CNPs co-express caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog or FGF2 signalling, efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally-specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from hESC include FGF8, canonical WNT and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube.
The great potential of induced pluripotent cells (iPS) cells is that it allows the possibility of... more The great potential of induced pluripotent cells (iPS) cells is that it allows the possibility of deriving pluripotent stem cells from any human patient. Generation of patient-derived stem cells serves as a great source for developing cell replacement therapies and also for creating human cellular model systems of specific diseases or disorders. This is only of benefit if there are well-established differentiation assay systems to generate the cell types of interest. This chapter describes robust and well-characterized protocols for differentiating iPS cells to neural progenitors, neurons, glia and neural crest cells. These established assays can be applied to iPS cell lines derived from patients with neurodegenerative disorders to study cellular mechanisms associated with neurodegeneration as well as investigating the regenerative potential of patient derived stem cells.
Stem cells are specialized cells that possess a capacity to undergo self-renewal while at the sam... more Stem cells are specialized cells that possess a capacity to undergo self-renewal while at the same time having the ability to give rise to at least one or more differentiated or mature cell type. They therefore represent a fundamental cornerstone during the life of all vertebrates, playing central roles in the production of new and replacement cells for tissues during development and homeostasis, including repair following disease or injury. This unit is a review of stem cells, their roles in development, and their potentials as therapeutic agents.
With the discovery two decades ago that the adult brain contains neural stem cells (NSCs) capable... more With the discovery two decades ago that the adult brain contains neural stem cells (NSCs) capable of producing new neurons, a great deal of research has been undertaken to manipulate these cells to repair the damaged nervous system. Much progress has been made in understanding what regulates adult neural stem cell specification, proliferation and differentiation but much remains to be determined. Lessons can be learned from understanding how embryonic neural stem cells produce the exquisitely complicated organ that is the adult mammalian nervous system. This review will highlight the role of transcriptional regulation of mammalian neural stem cells during embryonic development and compare these to the adult neural stem cell/neural precursor cell (NPC) niches of the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampal dentate gyrus. Normal physiological NSC/NPC regulation will be explored, as well as their regulation and responses following neural injury and disease. Finally, transcriptional regulation of the endogenous NSC/NPCs will be compared and contrasted with embryonic stem/induced pluripotent stem (ES/iPS) cell-derived NSC/NPCs. Recapitulation of the embryonic sequence of transcriptional events in neural stem cell development into specific neuronal or glial lineages improves directed differentiation of ES/iPS cells and may be useful for activation and specification of endogenous adult neural stem cells for therapeutic purposes.
Neural differentiation from embryonic stem cells involves progressive stages of neural induction,... more Neural differentiation from embryonic stem cells involves progressive stages of neural induction, expansion and maintenance of neural stem/progenitor cells, and differentiation to neurons and glia. Our understanding of the signals involved in each of these processes is primarily based on our knowledge of neural development during embryogenesis. This review will focus on the signalling pathways that have been identified to play a role in neural differentiation of human embryonic stem cells (hESCs), including their induction to neuroectoderm, maintenance and expansion of hESC-derived neurospheres, differentiation to neurons and specification to specific neuronal lineages. Understanding the signals involved in each of these stages is important for optimising methods to derive specific cell types for transplantation therapies, as well as for providing insight into the mechanisms of human neurogenesis.
The reported pluripotential capabilities of many human stem cell types has made them an attractiv... more The reported pluripotential capabilities of many human stem cell types has made them an attractive area of research, given the belief they may hold considerable therapeutic potential for treating a wide range of human diseases and injuries. Although the bulk of stem cell based research has focused on developing procedures for the treatment of pancreatic, neural, cardiovascular and haematopoietic diseases, the potential for deriving respiratory cell types from stem cells for treatment of respiratory specific diseases has also been explored. It is suggested that stem cell derivatives may be used for lung replacement/regeneration therapeutics and high though-put pharmacological screening strategies for a variety of respiratory injuries and diseases including: cystic fibrosis, chronic obstructive pulmonary disease, respiratory distress syndrome, pulmonary fibrosis and pulmonary edema. This review will explore recent progress in characterizing adult respiratory and bone marrow derived stem cells with respiratory potential as well as the endogenous mechanisms directing the homing of these cells to the diseased and injured lung. In addition, the potential for embryonic stem cell based therapies in this domain as well as the histological, anatomical and molecular aspects of respiratory development will be summarized.
Human embryonic stemlike cells (hESCs) are pluripotent cells derived from blastocysts. Differenti... more Human embryonic stemlike cells (hESCs) are pluripotent cells derived from blastocysts. Differentiating hESCs into respiratory lineages may benefit respiratory therapeutic programs. We previously demonstrated that 24% of all mouse embryonic stem cell (mESC) derivatives cocultured with embryonic day 11.5 (E11.5) mouse lung rudiments display immunoreactivity to the pneumonocyte II specific marker surfactant-associated protein C (Sftpc). Here we further investigate the effects of this inductive niche in terms of its competence to induce hESC derivative SFTPC immunoreactivity and the expression of other markers of terminal lung secretory units. When hESCs were cocultured as single cells, clumps of approximately 10 cells or embryoid bodies (EBs), hESC derivatives formed pan-keratin-positive epithelial tubules at high frequency (>30% of all hESC derivatives). However, human-specific SFTPC immunoreactivity associated with tubule formation only at low frequency (<0.1% of all hESC derivatives). Human-specific SFTPD and secretoglobin family 1A member 1 (SCGB1A1, also known as CC10) transcripts were detected by PCR after prolonged culture. Expression of other terminal lung secretory unit markers (TITF1, SFTPA, and SFTPB) was not detected at any time point analyzed. On the other hand, hESC derivatives cultured as plated EBs in media previously demonstrated to induce Sftpc expression in isolated mouse fetal tracheal epithelium expressed all terminal lung secretory unit markers examined. mESCs and hESCs thus display fundamental differences in their response to the E11.5 mouse lung inductive niche, and these data provide an important step in the delineation of signaling mechanisms capable of efficiently inducing hESC differentiation into terminal secretory units of the lung.
Uploads
Papers by Mark Denham