In Vitro Differentiation of Size‐Sieved Stem Cells into Electrically Active Neural Cells

Hung, Shih Chieh; Cheng, Henrich; Pan, Chien‐Yuan; Tsai, Ming-Tzu; Kao, Lung‐Sen; Li, Hsiao

doi:10.1634/stemcells.20-6-522

Cited by 138 publications

(109 citation statements)

References 32 publications

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“…We have previously investigated the signaling pathways involved in chondrogenesis under the control of type I and type II collagen, TGF␤1, and platelet-rich plasma (3,4,32,33,38). Building on the observations from our previous studies, we established a coculture system in which MSCs were directed toward chondrogenesis under the influence of mature chondrocytes, thereby creating a better simulation of physiologic conditions.…”

Section: Discussionmentioning

confidence: 99%

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In vitro stage‐specific chondrogenesis of mesenchymal stem cells committed to chondrocytes

Chen

Lai

et al. 2009

Arthritis & Rheumatism

110

118

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Objective. Osteoarthritis is characterized by an imbalance in cartilage homeostasis, which could potentially be corrected by mesenchymal stem cell (MSC)-based therapies. However, in vivo implantation of undifferentiated MSCs has led to unexpected results. This study was undertaken to establish a model for preconditioning of MSCs toward chondrogenesis as a more effective clinical tool for cartilage regeneration.Methods. A coculture preconditioning system was used to improve the chondrogenic potential of human MSCs and to study the detailed stages of chondrogenesis of MSCs, using a human MSC line, Kp-hMSC, in commitment cocultures with a human chondrocyte line, hPi (labeled with green fluorescent protein [GFP]). In addition, committed MSCs were seeded into a collagen scaffold and analyzed for their neocartilage-forming ability.Results. Coculture of hPi-GFP chondrocytes with Kp-hMSCs induced chondrogenesis, as indicated by the increased expression of chondrogenic genes and accumulation of chondrogenic matrix, but with no effect on osteogenic markers. The chondrogenic process of committed MSCs was initiated with highly activated chondrogenic adhesion molecules and stimulated cartilage developmental growth factors, including members of the transforming growth factor ␤ superfamily and their downstream regulators, the Smads, as well as endothelial growth factor, fibroblast growth factor, insulin-like growth factor, and vascular endothelial growth factor. Furthermore, committed Kp-hMSCs acquired neocartilageforming potential within the collagen scaffold. Osteoarthritis (OA), one of the most common musculoskeletal diseases, has been ascribed to an imbalance in cartilage homeostasis in the aging process (1,2). Hyaline cartilage has little capacity for self-repair. As a result, continuous mechanical stress can lead to the degradation of articular cartilage, culminating in a vicious cycle of destructive processes (3). Many therapeutic interventions directed at the restoration of the reparative capacity of chondrocytes have been explored (3-5). One of the emerging approaches is cell-based therapy, in which expanded chondrocytes are used for cartilage repair. However, the outcome of this approach has been disappointing, due to the difficulties in maintaining chondrocytic phenotypes and the decrease in proliferative capacity of autologous chondrocytes with increasing age (1,6,7). Conclusion. These findings help define the molecular markers of chondrogenesis and more accuratelyCell therapy based on autologous mesenchymal stem cells (MSCs), which have a vast proliferative ca-

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Section: Discussionmentioning

confidence: 99%

“…We have previously investigated the methods of purification, characterization, differentiation, and application of multipotent MSCs (10,(32)(33)(34)(35). We have also studied the expression of matrix proteins, the type I and type II collagens, which induce chondrogenesis in human MSCs under the influence of TGF␤1 (3).…”

mentioning

confidence: 99%

In vitro stage‐specific chondrogenesis of mesenchymal stem cells committed to chondrocytes

Chen

Lai

et al. 2009

Arthritis & Rheumatism

110

118

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“…hMSCs can be induced to differentiate into multiple lineages, including bone, fat, cartilage, as well as neuron in vitro (1,(3)(4)(5). Transplanted MSCs are able to differentiate into neuronal lineage and improve the functions of central nervous system (CNS) with ischemia (6), Parkinson disease (7), Alzheimer disease (8), spinal cord injury (9, 10), and other neurodegenerative disorders (11, 12) modeled in rodents.…”

Section: Human Mesenchymal Stem Cells (Hmscs)mentioning

confidence: 99%

EZH2 Regulates Neuronal Differentiation of Mesenchymal Stem Cells through PIP5K1C-dependent Calcium Signaling

Chou

Chen

et al. 2011

Journal of Biological Chemistry

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Enhancer of zeste homolog 2 (EZH2) regulates stem cells renewal, maintenance, and differentiation into different cell lineages including neuron. Changes in intracellular Ca2؉ concentration play a critical role in the differentiation of neurons. However, whether EZH2 modulates intracellular Ca 2؉ signaling in regulating neuronal differentiation from human mesenchymal stem cells (hMSCs) still remains unclear. When hMSCs were treated with a Ca 2؉ chelator or a PLC inhibitor to block IP 3 -mediated Ca 2؉ signaling, neuronal differentiation was disrupted. EZH2 bound to the promoter region of PIP5K1C to suppress its transcription in proliferating hMSCs. Interestingly, knockdown of EZH2 enhanced the expression of PIP5K1C, which in turn increased the amount of PI(4,5)P 2 , a precursor of IP 3 , and resulted in increasing the intracellular Ca 2؉ level, suggesting that EZH2 negatively regulates intracellular Ca 2؉ through suppression of PIP5K1C. Knockdown of EZH2 also enhanced hMSCs differentiation into functional neuron both in vitro and in vivo. In contrast, knockdown of PIP5K1C significantly reduced PI(4,5)P 2 contents and intracellular Ca 2؉ release in EZH2-silenced cells and resulted in the disruption of neuronal differentiation from hMSCs. Here, we provide the first evidence to demonstrate that after induction to neuronal differentiation, decreased EZH2 activates the expression of PIP5K1C to evoke intracellular Ca 2؉ signaling, which leads hMSCs to differentiate into functional neuron lineage. Activation of intracellular Ca 2؉ signaling by repressing or knocking down EZH2 might be a potential strategy to promote neuronal differentiation from hMSCs for application to neurological dysfunction diseases. Human mesenchymal stem cells (hMSCs)4 derived from bone marrow are easily obtained (1), safely expanded in vitro and are not susceptible to malignant transformation; thus, hMSCs are suitable for therapeutic applications (2). hMSCs can be induced to differentiate into multiple lineages, including bone, fat, cartilage, as well as neuron in vitro (1,(3)(4)(5). Transplanted MSCs are able to differentiate into neuronal lineage and improve the functions of central nervous system (CNS) with ischemia (6), Parkinson disease (7), Alzheimer disease (8), spinal cord injury (9, 10), and other neurodegenerative disorders (11, 12) modeled in rodents. These findings demonstrate the potential of hMSCs for cell therapy in human CNS.Spontaneous and transient elevation in intracellular Ca 2ϩ concentration during an early period is required and critical for neuronal differentiation both in vitro and in embryonic neuron development (13). However, whether intracellular Ca 2ϩ signaling is required for neuronal differentiation from hMSCs remains unclear. The expression of transient receptor potential (TRP) proteins, TRPC1 and TRPC3, is elevated to activate store-operated calcium entry (SOCE) after differentiation of H19 -7 hippocampal neuronal cells (14). It is also known that inositol 1,4,5-trisphosphate (IP 3 ) stimulates a ligand-gated chann...

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“…However, the density of the voltage-gated sodium channels is very low compared to mature neurons, suggesting that the undifferentiated hMeSCs are incapable of producing action potentials. Attempts to induce action potentials in hMeSCs using depolarizing current pulses have confirmed that undifferentiated MeSCs isolated from the adult BM of humans or rodents do not generate action potentials [14,21,25,[37][38][39][40].…”

Section: Membrane Properties Of Neuron-like Cells Differentiated Frommentioning

confidence: 99%

“…Previous calcium imaging studies have show that cells differentiated from adult MeSCs, adult NSCs, and embryonic stem cells respond to depolarization with KCl or the application of the neurotransmitter l-glutamate with an increase in cytoplasmic Ca 2þ levels [27,31,38,55,56]. These transient changes in the Ca 2þ levels are important for regulating neuronal differentiation, transmitter selection, and axonal targeting in immature neurons [57,58].…”

Section: Development Of Kcl and L-glutamate-induced Increases In Cytomentioning

confidence: 99%

Membrane Properties of Neuron-Like Cells Generated from Adult Human Bone-Marrow-Derived Mesenchymal Stem Cells

Fox

Shen

et al. 2010

Stem Cells and Development

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Adult mesenchymal stem cells (MeSCs) isolated from human bone marrow are capable of generating neural stem cell (NSC)-like cells that can be subsequently differentiated into cells expressing molecular markers for neurons. Here we report that these neuron-like cells had functional properties similar to those of brain-derived neurons. Whole-cell patch-clamp recordings and calcium imaging experiments were performed on neuron-like cells differentiated from bone-marrow-derived NSC-like cells. The neuron-like cells were subjected to current pulses to determine if they were capable of generating depolarization-induced action potentials. We found that nearly all of the cells with neuron-like morphology exhibited active membrane properties in response to the depolarizing pulses. The most common response was a single spike-like event with an overshoot and brief afterhyperpolarization. Cells that did not generate overshooting spike-like events usually displayed rectifying current-voltage relationships. The prevalence of these active membrane properties in response to the depolarizing current pulses suggested that the human MeSCs (hMeSCs) were capable of converting to a neural lineage under defined culture conditions. The spike-like events were blocked by the voltage-gated sodium channel inhibitor lidocaine, but unaffected by another sodium channel inhibitor tetrodotoxin (TTX). In calcium imaging experiments, the neuron-like cells responded to potassium chloride depolarization and l-glutamate application with increases in the cytoplasmic calcium levels. Thus, the neuron-like cells appeared to express TTX-resistant voltage-gated sodium channels, voltage-gated calcium channels, and functional l-glutamate receptors. These results demonstrate that hMeSCs were capable of generating cells with characteristics typical of functional neurons that may prove useful for neuroreplacement therapies.

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In Vitro Differentiation of Size‐Sieved Stem Cells into Electrically Active Neural Cells

Cited by 138 publications

References 32 publications

In vitro stage‐specific chondrogenesis of mesenchymal stem cells committed to chondrocytes

In vitro stage‐specific chondrogenesis of mesenchymal stem cells committed to chondrocytes

EZH2 Regulates Neuronal Differentiation of Mesenchymal Stem Cells through PIP5K1C-dependent Calcium Signaling

Membrane Properties of Neuron-Like Cells Generated from Adult Human Bone-Marrow-Derived Mesenchymal Stem Cells

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