Induction of hematoendothelial and neurovascular lineages using human pluripotent stem cells

The regenerative capabilities of human pluripotent stem cells (hPSCs) have transformed the landscape of translational research, providing alternative approaches to treating devastating diseases. hPSCs are defined by the ability to self-renew and differentiate into all three germ layers: ectoderm, mesoderm, and endoderm, and have the potential to give rise to any cell type in the human body. The purpose of this research is to harness the differentiation potential of hPSCs to better understand the development of mesodermal and neural crest-derived blood-brain barrier pericytes of the central nervous system (CNS) and mesodermal-derived hematopoietic stem cells (HSCs) that give rise to the blood and immune systems. A single HSC has the potential to give rise to the entire blood system. However, the induction of definitive hematopoietic progenitors, which display multipotency and can home and engraft to the bone marrow upon transplantation, has proven difficult. Thus, the goal of this project is to develop a differentiation protocol to generate definitive hematopoietic progenitors from hPSCs. This work was later adapted to make neurovascular cells of the CNS, with a particular focus on pericytes.Pericytes are a mural cell found in close association with endothelial cells and are essential to vascular maturation and maintenance. Pericytes have diverse developmental origins and are found in a wide variety of tissues and organs throughout the body. In particular, pericytes are a major component of the blood-brain barrier (BBB), which is also comprised of endothelial cells and astrocytes. The BBB is a selectively permeable network of blood vessels that protects and maintains homeostasis of the central nervous system (CNS) by regulating the transportation of molecules into and out of the brain. Dysfunction of the BBB has been implicated in the progression of Alzheimer’s Disease (AD), in which the accumulation of neurotoxic plaques of beta amyloid (Aβ) peptides leads to neuronal loss, cognitive decline, and, ultimately, death. However, the mechanism of BBB breakdown in AD, including the disrupted interactions between the three cell types, are not well understood. While protocols to generate astrocytes and brain endothelial cells from hPSCs exist, there are currently no protocols to create iPSC-derived CNS-specific pericytes. The primary focus of this work is to generate brain-specific pericytes from induced pluripotent stem cell (iPSC) lines created from patients bearing alleles for APOE3 or APOE4, which is the single greatest genetic risk factor for AD

Therapeutic response of GBM stem-like cells and the GBM cell of origin

Includes bibliographical references (leaves 61-67)Glioblastoma Multiforme (GBM) is the most common and aggressive type of adult brain cancer that offers only one to two years of survival and results in the growth of brain tumors. GBM was studied by a group of researchers called The Cancer Genome Atlas (TCGA). This group identified four distinct types of GBM that differ from each other based on unique genetic mutations, rate of tumor growth, and response to treatment. Some cancer cells have stem cell properties such as the ability to give rise to multiple cell types and maintenance of self-renewal, which may advance the rate of tumor growth. It is believed that this cancer stem cell population infers resistance to radiation and Temozolomide (TMZ) chemotherapeutic treatment in GBM. TMZ eradicates cells by alkylating their DNA to cause cell death. Working with primary patient derived GBM stem-like cell cultures, we show that the cultures separate into different response groups that will be used in later studies to classify the cultures into TCGA subgroups. Additionally, we also show that expression of the DNA repair enzyme, MGMT, which removes TMZ induced DNA alkylation, infers GBM culture resistance to TMZ.\ud \ud Though much work has been conducted on the genetic anomalies prevalent and efficacy of treatment of GBM, little is known about the cell of origin in GBM. In fact, the cell of origin for GBM has not yet been identified. The main purpose of the second portion of this research project is to determine the role genetic mutations in the tumor suppressor gene, PTEN, and the oncogenic gene, KRAS, in specific brain cells and their potential to produce GBM tumors in mouse brains. We hypothesize that loss of PTEN and over expression of KRAS in NG2, which is a proteoglycan found on neural progenitor cells, positive mouse cells will result in tumor formation. A CRE-LOX breeding system was used to successfully generate genetic mutations in the mice. We have successfully bred mice that express Pten and contain Kras genetic mutations. We are currently conducting the final genetic cross to determine whether tumors will form from the NG2 positive cells. \ud \ud We have successfully identified groups of our GBM neursophere cultures that differ from each other based on their response to treatment. This data will be used when we begin to classify our cultures into one of the four TCGA GBM subgroups. Additionally, identifying the cell type of origin for GBM will also provide important insight on how to combat this cancer to develop more targeted cancer therapy in the future

Induction of Mesoderm and Neural Crest-Derived Pericytes from Human Pluripotent Stem Cells to Study Blood-Brain Barrier Interactions

Summary: In the CNS, perivascular cells (“pericytes”) associate with endothelial cells to mediate the formation of tight junctions essential to the function of the blood-brain barrier (BBB). The BBB protects the CNS by regulating the flow of nutrients and toxins into and out of the brain. BBB dysfunction has been implicated in the progression of Alzheimer's disease (AD), but the role of pericytes in BBB dysfunction in AD is not well understood. In the developing embryo, CNS pericytes originate from two sources: mesoderm and neural crest. In this study, we report two protocols using mesoderm or neural crest intermediates, to generate brain-specific pericyte-like cells from induced pluripotent stem cell (iPSC) lines created from healthy and AD patients. iPSC-derived pericytes display stable expression of pericyte surface markers and brain-specific genes and are functionally capable of increasing vascular tube formation and endothelial barrier properties. : While protocols to generate pericytes from hPSC lines exist, differentiation of brain-specific pericytes has not been reported. In this article, Faal and colleagues developed two robust and highly scalable methods relying on either mesoderm or neural crest induction to generate brain pericyte-like cells from hPSCs. Resulting cells express pericyte markers and brain-specific genes and improve barrier quality of endothelial cells. Keywords: pericytes, endothelial cells, human pluripotent stem cells, mesoderm, neural crest, blood-brain barrier, Alzheimer's diseas

Induction of Mesoderm and Neural Crest-Derived Pericytes from Human Pluripotent Stem Cells to Study Blood-Brain Barrier Interactions.

In the CNS, perivascular cells ("pericytes") associate with endothelial cells to mediate the formation of tight junctions essential to the function of the blood-brain barrier (BBB). The BBB protects the CNS by regulating the flow of nutrients and toxins into and out of the brain. BBB dysfunction has been implicated in the progression of Alzheimer's disease (AD), but the role of pericytes in BBB dysfunction in AD is not well understood. In the developing embryo, CNS pericytes originate from two sources: mesoderm and neural crest. In this study, we report two protocols using mesoderm or neural crest intermediates, to generate brain-specific pericyte-like cells from induced pluripotent stem cell (iPSC) lines created from healthy and AD patients. iPSC-derived pericytes display stable expression of pericyte surface markers and brain-specific genes and are functionally capable of increasing vascular tube formation and endothelial barrier properties

4-Hydroxytamoxifen probes for light-dependent spatiotemporal control of Cre-ER mediated reporter gene expression.

The tamoxifen inducible Cre-ER/loxP system provides tissue specific temporal control of gene recombination events, and can be used to induce expression of reporter genes (e.g. GFP, LacZ) for lineage tracing studies. Cre enzyme fused with estrogen receptor (Cre-ER) is released upon tamoxifen binding, resulting in permanent activation of reporter genes within cells and their progeny. Tamoxifen and its active metabolite, hydroxytamoxifen (4OHT) diffuses rapidly in vivo, making it difficult to restrict labeling to specific locations. In this study, we developed a photocaged 4OHT molecule by covalently attaching 4OHT to an ortho-nitrobenzyl (ONB1) group, rendering 4OHT inactive. Exposure to UV radiation cleaves the bond between ONB1 and 4OHT, freeing the 4OHT to bind Cre-ER to result in downstream genetic recombination and reporter activation. We show that caged ONB1-4OHT crosses the cell membrane and uncages after short UV exposure, resulting in Cre-driven genetic recombination that can be localized to specific regions or tissues. ONB1-4OHT can provide spatial control of reporter activation and be adapted with any existing Cre-ER/loxP based system

4-Hydroxytamoxifen probes for light-dependent spatiotemporal control of Cre-ER mediated reporter gene expression

The tamoxifen inducible Cre-ER/loxP system provides tissue specific temporal control of gene recombination events, and can be used to induce expression of reporter genes (e.g. GFP, LacZ) for lineage tracing studies. Cre enzyme fused with estrogen receptor (Cre-ER) is released upon tamoxifen binding, resulting in permanent activation of reporter genes within cells and their progeny. Tamoxifen and its active metabolite, hydroxytamoxifen (4OHT) diffuses rapidly in vivo, making it difficult to restrict labeling to specific locations. In this study, we developed a photocaged 4OHT molecule by covalently attaching 4OHT to an ortho-nitrobenzyl (ONB1) group, rendering 4OHT inactive. Exposure to UV radiation cleaves the bond between ONB1 and 4OHT, freeing the 4OHT to bind Cre-ER to result in downstream genetic recombination and reporter activation. We show that caged ONB1-4OHT crosses the cell membrane and uncages after short UV exposure, resulting in Cre-driven genetic recombination that can be localized to specific regions or tissues. ONB1-4OHT can provide spatial control of reporter activation and be adapted with any existing Cre-ER/loxP based system