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Reprogramming of HUVECs into induced pluripotent stem cells (HiPSCs), generation and characterization of HiPSC-derived neurons and astrocytes.

Haile Y, Nakhaei-Nejad M, Boakye PA, Baker G, Smith PA, Murray AG, Giuliani F, Jahroudi N - PLoS ONE (2015)

Bottom Line: Limitations related to the animal models of these human diseases have impeded the development of effective drugs.HiPSC-derived neurons possess similar morphology but significantly longer neurites compared to primary human fetal neurons.In summary, this study presents a novel technique to generate differentiated and functional HiPSC-derived neurons and astrocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Alberta, Edmonton, Canada.

ABSTRACT
Neurodegenerative diseases are characterized by chronic and progressive structural or functional loss of neurons. Limitations related to the animal models of these human diseases have impeded the development of effective drugs. This emphasizes the need to establish disease models using human-derived cells. The discovery of induced pluripotent stem cell (iPSC) technology has provided novel opportunities in disease modeling, drug development, screening, and the potential for "patient-matched" cellular therapies in neurodegenerative diseases. In this study, with the objective of establishing reliable tools to study neurodegenerative diseases, we reprogrammed human umbilical vein endothelial cells (HUVECs) into iPSCs (HiPSCs). Using a novel and direct approach, HiPSCs were differentiated into cells of central nervous system (CNS) lineage, including neuronal, astrocyte and glial cells, with high efficiency. HiPSCs expressed embryonic genes such as nanog, sox2 and Oct-3/4, and formed embryoid bodies that expressed markers of the 3 germ layers. Expression of endothelial-specific genes was not detected in HiPSCs at RNA or protein levels. HiPSC-derived neurons possess similar morphology but significantly longer neurites compared to primary human fetal neurons. These stem cell-derived neurons are susceptible to inflammatory cell-mediated neuronal injury. HiPSC-derived neurons express various amino acids that are important for normal function in the CNS. They have functional receptors for a variety of neurotransmitters such as glutamate and acetylcholine. HiPSC-derived astrocytes respond to ATP and acetylcholine by elevating cytosolic Ca2+ concentrations. In summary, this study presents a novel technique to generate differentiated and functional HiPSC-derived neurons and astrocytes. These cells are appropriate tools for studying the development of the nervous system, the pathophysiology of various neurodegenerative diseases and the development of potential drugs for their treatments.

No MeSH data available.


Related in: MedlinePlus

Feeder-layer independent induced pluripotent stem cells generation from HUVECs (HiPSCs).(A) HUVECs were transduced with lentiviral vectors. Within 4 days (B) and a week (C) the endothelial cells were reduced in density but formed aggregates. (D) On day 16, immature colonies emerged. (E and F) Fully reprogrammed human embryonic stem cells (hESC)-like colonies were isolated after 21 days. Micrographs (G and I in low magnification) and (H and J in high magnification) show the morphology and quality of the colonies. Scale bars: A-F = 200 μm, G and I = 100 μm, H and J = 400 μm.
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pone.0119617.g001: Feeder-layer independent induced pluripotent stem cells generation from HUVECs (HiPSCs).(A) HUVECs were transduced with lentiviral vectors. Within 4 days (B) and a week (C) the endothelial cells were reduced in density but formed aggregates. (D) On day 16, immature colonies emerged. (E and F) Fully reprogrammed human embryonic stem cells (hESC)-like colonies were isolated after 21 days. Micrographs (G and I in low magnification) and (H and J in high magnification) show the morphology and quality of the colonies. Scale bars: A-F = 200 μm, G and I = 100 μm, H and J = 400 μm.

Mentions: HUVEC-derived iPSCs (HiPSCs), using feeder-layers, have been established and are well characterized [14–16]. Since contamination of iPSCs with animal cells is a major limitation for potential therapeutic purposes, we aimed to generate iPSCs from HUVECs independent of feeder layers. Using the protocol described in the Materials and Methods, HUVECs were transduced with lentiviral vectors expressing reprogramming transcription factors. Transduction efficiency was 78–80%, determined based on GFP expression by cells that were independently transduced with a lentivirus vector expressing GFP. Four days post transduction, the HUVEC monolayer (Fig. 1A) started to reduce density (Fig. 1B). Within a week, the density of the cells was reduced and aggregated cells started to form (Fig. 1C). On day 16, colonies emerged (Fig. 1D), and on day 20, fully reprogrammed colonies were formed (Fig. 1E and F). The morphology of the colonies was typical for iPSCs [31] and showed a well defined edge composed of tightly packed round and uniformly sized cells (Fig. 1G, I in low magnification; and H, J in high magnification).


Reprogramming of HUVECs into induced pluripotent stem cells (HiPSCs), generation and characterization of HiPSC-derived neurons and astrocytes.

Haile Y, Nakhaei-Nejad M, Boakye PA, Baker G, Smith PA, Murray AG, Giuliani F, Jahroudi N - PLoS ONE (2015)

Feeder-layer independent induced pluripotent stem cells generation from HUVECs (HiPSCs).(A) HUVECs were transduced with lentiviral vectors. Within 4 days (B) and a week (C) the endothelial cells were reduced in density but formed aggregates. (D) On day 16, immature colonies emerged. (E and F) Fully reprogrammed human embryonic stem cells (hESC)-like colonies were isolated after 21 days. Micrographs (G and I in low magnification) and (H and J in high magnification) show the morphology and quality of the colonies. Scale bars: A-F = 200 μm, G and I = 100 μm, H and J = 400 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4366250&req=5

pone.0119617.g001: Feeder-layer independent induced pluripotent stem cells generation from HUVECs (HiPSCs).(A) HUVECs were transduced with lentiviral vectors. Within 4 days (B) and a week (C) the endothelial cells were reduced in density but formed aggregates. (D) On day 16, immature colonies emerged. (E and F) Fully reprogrammed human embryonic stem cells (hESC)-like colonies were isolated after 21 days. Micrographs (G and I in low magnification) and (H and J in high magnification) show the morphology and quality of the colonies. Scale bars: A-F = 200 μm, G and I = 100 μm, H and J = 400 μm.
Mentions: HUVEC-derived iPSCs (HiPSCs), using feeder-layers, have been established and are well characterized [14–16]. Since contamination of iPSCs with animal cells is a major limitation for potential therapeutic purposes, we aimed to generate iPSCs from HUVECs independent of feeder layers. Using the protocol described in the Materials and Methods, HUVECs were transduced with lentiviral vectors expressing reprogramming transcription factors. Transduction efficiency was 78–80%, determined based on GFP expression by cells that were independently transduced with a lentivirus vector expressing GFP. Four days post transduction, the HUVEC monolayer (Fig. 1A) started to reduce density (Fig. 1B). Within a week, the density of the cells was reduced and aggregated cells started to form (Fig. 1C). On day 16, colonies emerged (Fig. 1D), and on day 20, fully reprogrammed colonies were formed (Fig. 1E and F). The morphology of the colonies was typical for iPSCs [31] and showed a well defined edge composed of tightly packed round and uniformly sized cells (Fig. 1G, I in low magnification; and H, J in high magnification).

Bottom Line: Limitations related to the animal models of these human diseases have impeded the development of effective drugs.HiPSC-derived neurons possess similar morphology but significantly longer neurites compared to primary human fetal neurons.In summary, this study presents a novel technique to generate differentiated and functional HiPSC-derived neurons and astrocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Alberta, Edmonton, Canada.

ABSTRACT
Neurodegenerative diseases are characterized by chronic and progressive structural or functional loss of neurons. Limitations related to the animal models of these human diseases have impeded the development of effective drugs. This emphasizes the need to establish disease models using human-derived cells. The discovery of induced pluripotent stem cell (iPSC) technology has provided novel opportunities in disease modeling, drug development, screening, and the potential for "patient-matched" cellular therapies in neurodegenerative diseases. In this study, with the objective of establishing reliable tools to study neurodegenerative diseases, we reprogrammed human umbilical vein endothelial cells (HUVECs) into iPSCs (HiPSCs). Using a novel and direct approach, HiPSCs were differentiated into cells of central nervous system (CNS) lineage, including neuronal, astrocyte and glial cells, with high efficiency. HiPSCs expressed embryonic genes such as nanog, sox2 and Oct-3/4, and formed embryoid bodies that expressed markers of the 3 germ layers. Expression of endothelial-specific genes was not detected in HiPSCs at RNA or protein levels. HiPSC-derived neurons possess similar morphology but significantly longer neurites compared to primary human fetal neurons. These stem cell-derived neurons are susceptible to inflammatory cell-mediated neuronal injury. HiPSC-derived neurons express various amino acids that are important for normal function in the CNS. They have functional receptors for a variety of neurotransmitters such as glutamate and acetylcholine. HiPSC-derived astrocytes respond to ATP and acetylcholine by elevating cytosolic Ca2+ concentrations. In summary, this study presents a novel technique to generate differentiated and functional HiPSC-derived neurons and astrocytes. These cells are appropriate tools for studying the development of the nervous system, the pathophysiology of various neurodegenerative diseases and the development of potential drugs for their treatments.

No MeSH data available.


Related in: MedlinePlus