Optimization of Long-Term Human iPSC-Derived Spinal Motor Neuron Culture Using a Dendritic Polyglycerol Amine-Based Substrate

Human induced pluripotent stem cells (hiPSCs) derived from healthy and diseased individuals can give rise to many cell types, facilitating the study of mechanisms of development, human disease modeling, and early drug target validation. In this context, experimental model systems based on hiPSC-derived motor neurons (MNs) have been used to study MN diseases such as spinal muscular atrophy and amyotrophic lateral sclerosis. Modeling MN disease using hiPSC-based approaches requires culture conditions that can recapitulate in a dish the events underlying differentiation, maturation, aging, and death of MNs. Current hiPSC-derived MN-based applications are often hampered by limitations in our ability to monitor MN morphology, survival, and other functional properties over a prolonged timeframe, underscoring the need for improved long-term culture conditions. Here we describe a cytocompatible dendritic polyglycerol amine (dPGA) substrate-based method for prolonged culture of hiPSC-derived MNs. We provide evidence that MNs cultured on dPGA-coated dishes are more amenable to long-term study of cell viability, molecular identity, and spontaneous network electrophysiological activity. The present study has the potential to improve hiPSC-based studies of human MN biology and disease. We describe the use of a new coating substrate providing improved conditions for long-term cultures of human iPSC-derived motor neurons, thus allowing evaluation of cell viability, molecular identity, spontaneous network electrophysiological activity, and single-cell RNA sequencing of mature motor neurons.

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Optimization of long-term human iPSC-derived spinal motor neuron culture using a dendritic polyglycerol amine-based substrate

10.1101/2021.09.14.460098 ◽

2021 ◽

Author(s):

Louise Thiry ◽

Jean-Pierre Clement ◽

Rainer Haag ◽

Timothy E Kennedy ◽

Stefano Stifani

Keyword(s):

Motor Neurons ◽

Disease Modeling ◽

Muscular Atrophy ◽

Cell Types ◽

Culture Conditions ◽

Model Systems ◽

Neuron Culture ◽

Long Term Study ◽

Drug Target Validation

Human induced pluripotent stem cells (h-iPSCs) derived from healthy and diseased individuals can give rise to many cell types, facilitating the study of mechanisms of development, human disease modeling, and early drug target validation. In this context, experimental model systems based on h-iPSC-derived motor neurons (MNs) have been used to study MN diseases such as spinal muscular atrophy and amyotrophic lateral sclerosis. Modeling MN disease using h-iPSC-based approaches requires culture conditions that can recapitulate in a dish the events underlying differentiation, maturation, aging, and death of MNs. Current h-iPSC-derived MN-based applications are often hampered by limitations in our ability to monitor MN morphology, survival, and other functional properties over a prolonged timeframe, underscoring the need for improved long-term culture conditions. Here we describe a cytocompatible dendritic polyglycerol amine (dPGA) substrate-based method for prolonged culture of h-iPSC-derived MNs. We provide evidence that MNs cultured on dPGA-coated dishes are more amenable to long-term study of cell viability, molecular identity, and spontaneous network electrophysiological activity. The present study has the potential to improve iPSC-based studies of human MN biology and disease.

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Long-term In Vitro Toxicity Models: Comparisons Between a Flow-cell Bioreactor, a Static-cell Bioreactor and Static Cell Cultures

Alternatives to Laboratory Animals ◽

10.1177/026119290203000505 ◽

2002 ◽

Vol 30 (5) ◽

pp. 515-523 ◽

Cited By ~ 13

Author(s):

Patricia Pazos ◽

Salvador Fortaner ◽

Pilar Prieto

Keyword(s):

Cell Culture ◽

Cell Viability ◽

Flow Cell ◽

Model Systems ◽

In Vitro Toxicity ◽

Protein Determination ◽

Efficient System ◽

Cytotoxicity Studies

In vitro long-term toxicity testing is becoming an important issue in the field of toxicology, and there is a need to develop new model systems that mimic human chronic exposure and its effects. The aim of this work was to test two long-term in vitro toxicity systems which are available, a flow-cell bioreactor (Tecnomouse) and a static cell bioreactor system (CELLine CL 6-well), and to compare them with the use of conventional cell culture flasks. A human cell line, Int 407, was exposed to cadmium chloride (CdCl2; 10–7–10–8M) for 4 weeks. Cell numbers and cell viabilities were determined by the trypan blue (TB) exclusion assay and from exclusion of propidium iodide (PI) as determined by flow cytometry; and cell viability and metabolic activity were determined by the MTT assay. In addition, total protein determination and cadmium uptake measurements were performed. The results obtained with TB and PI exclusion did not show clear differences in cell viability with increasing CdCl2 concentration. However, in the static cell-culture systems, an increase in MTT reduction was found at low concentrations of CdCl2. Expression of heat-shock protein (Hsp27 and Hsp70) increased differently, depending on the CdCl2 concentration applied and the system used. In summary, of the two bioreactors, the CELLine CL 6-well bioreactor was shown to be the more efficient system for performing long-term cytotoxicity studies. It is easy to handle, it permits the assessment of several endpoints, and sufficient replicates can be made available.

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Long-term single-cell passaging of human iPSC fully supports pluripotency and high-efficient trilineage differentiation capacity

10.1101/663047 ◽

2019 ◽

Cited By ~ 1

Author(s):

Estela Cruvinel ◽

Isabella Ogusuku ◽

Rosanna Cerioni ◽

Jéssica Gonçalves ◽

Maria Elisa Góes ◽

...

Keyword(s):

Single Cell ◽

Disease Modeling ◽

Embryoid Body ◽

Population Doubling ◽

Population Doubling Time ◽

Differentiation Capacity ◽

Culture Cells ◽

High Efficient ◽

Human Ipsc

ABSTRACTObjectivesTo validate a straightforward single-cell passaging cultivation method that enables high-quality maintenance of hiPSC without the appearance of karyotypic abnormalities or loss of pluripotency.MethodsCells were kept in culture for over 50 passages, following a structured chronogram of passage and monitoring cell growth by population doubling time (PDT) calculation and cell confluence. Standard procedures for iPSC monitoring as embryonic body (EB) formation, karyotyping and pluripotency markers expression were evaluated in order to monitor the cellular state in the long-term culture. Cells that underwent these tests were then subjected to differentiation into keratinocytes and cardiomyocytes to evaluate its differentiation capacity.ResultshiPSC clones maintained its pluripotent capability as well as chromosomal integrity and were able to generate derivatives from the three germ layers at high passages by embryoid body formation and high-efficient direct differentiation into keratinocytes and cardiomyocytes.ConclusionOur findings support the routine of hiPSC single-cell passaging as a reliable procedure even after long-term cultivation, providing healthy PSCs to be used in drugs discovery, toxicity and disease modeling as well as for therapeutic approaches.

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Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia

Nature Communications ◽

10.1038/s41467-021-25344-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Reina Bassil ◽

Kenneth Shields ◽

Kevin Granger ◽

Ivan Zein ◽

Shirley Ng ◽

...

Keyword(s):

Disease Modeling ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Induced Pluripotent Stem Cell ◽

Dystrophic Neurites ◽

Synapse Loss ◽

Induced Pluripotent ◽

Human Ipsc ◽

Dendrite Retraction

AbstractAdvancement in human induced pluripotent stem cell (iPSC) neuron and microglial differentiation protocols allow for disease modeling using physiologically relevant cells. However, iPSC differentiation and culturing protocols have posed challenges to maintaining consistency. Here, we generated an automated, consistent, and long-term culturing platform of human iPSC neurons, astrocytes, and microglia. Using this platform we generated a iPSC AD model using human derived cells, which showed signs of Aβ plaques, dystrophic neurites around plaques, synapse loss, dendrite retraction, axon fragmentation, phospho-Tau induction, and neuronal cell death in one model. We showed that the human iPSC microglia internalized and compacted Aβ to generate and surround the plaques, thereby conferring some neuroprotection. We investigated the mechanism of action of anti-Aβ antibodies protection and found that they protected neurons from these pathologies and were most effective before pTau induction. Taken together, these results suggest that this model can facilitate target discovery and drug development efforts.

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A Perfusion Bioreactor for Longitudinal Monitoring of Bioengineered Liver Constructs

Nanomaterials ◽

10.3390/nano11020275 ◽

2021 ◽

Vol 11 (2) ◽

pp. 275

Author(s):

Lisa Sassi ◽

Omolola Ajayi ◽

Sara Campinoti ◽

Dipa Natarajan ◽

Claire McQuitty ◽

...

Keyword(s):

Tissue Engineering ◽

Liver Disease ◽

Cell Viability ◽

3D Culture ◽

Culture Conditions ◽

Disease Models ◽

Non Invasive ◽

And Function

In the field of in vitro liver disease models, decellularised organ scaffolds maintain the original biomechanical and biological properties of the extracellular matrix and are established supports for in vitro cell culture. However, tissue engineering approaches based on whole organ decellularized scaffolds are hampered by the scarcity of appropriate bioreactors that provide controlled 3D culture conditions. Novel specific bioreactors are needed to support long-term culture of bioengineered constructs allowing non-invasive longitudinal monitoring. Here, we designed and validated a specific bioreactor for long-term 3D culture of whole liver constructs. Whole liver scaffolds were generated by perfusion decellularisation of rat livers. Scaffolds were seeded with Luc+HepG2 and primary human hepatocytes and cultured in static or dynamic conditions using the custom-made bioreactor. The bioreactor included a syringe pump, for continuous unidirectional flow, and a circuit built to allow non-invasive monitoring of culture parameters and media sampling. The bioreactor allowed non-invasive analysis of cell viability, distribution, and function of Luc+HepG2-bioengineered livers cultured for up to 11 days. Constructs cultured in dynamic conditions in the bioreactor showed significantly higher cell viability, measured with bioluminescence, distribution, and functionality (determined by albumin production and expression of CYP enzymes) in comparison to static culture conditions. Finally, our bioreactor supports primary human hepatocyte viability and function for up to 30 days, when seeded in the whole liver scaffolds. Overall, our novel bioreactor is capable of supporting cell survival and metabolism and is suitable for liver tissue engineering for the development of 3D liver disease models.

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Respecifying human iPSC-derived blood cells into highly engraftable hematopoietic stem and progenitor cells with a single factor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1718446115 ◽

2018 ◽

Vol 115 (9) ◽

pp. 2180-2185 ◽

Cited By ~ 23

Author(s):

Yu-Ting Tan ◽

Lin Ye ◽

Fei Xie ◽

Ashley I. Beyer ◽

Marcus O. Muench ◽

...

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Blood Cells ◽

Disease Modeling ◽

Single Factor ◽

Leukemic Transformation ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Human Ipsc

Derivation of human hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs) offers considerable promise for cell therapy, disease modeling, and drug screening. However, efficient derivation of functional iPSC-derived HSCs with in vivo engraftability and multilineage potential remains challenging. Here, we demonstrate a tractable approach for respecifying iPSC-derived blood cells into highly engraftable hematopoietic stem and progenitor cells (HSPCs) through transient expression of a single transcription factor, MLL-AF4. These induced HSPCs (iHSPCs) derived from iPSCs are able to fully reconstitute the human hematopoietic system in the recipient mice without myeloid bias. iHSPCs are long-term engraftable, but they are also prone to leukemic transformation during the long-term engraftment period. On the contrary, primary HSPCs with the same induction sustain the long-term engraftment without leukemic transformation. These findings demonstrate the feasibility of activating the HSC network in human iPSC-derived blood cells through expression of a single factor and suggest iHSPCs are more genomically instable than primary HSPCs, which merits further attention.

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Long-term iodination of thyroglobulin by porcine thyroid cells cultured in porous-bottomed culture chambers: regulation by thyrotrophin

Journal of Endocrinology ◽

10.1677/joe.0.1280051 ◽

1991 ◽

Vol 128 (1) ◽

pp. 51-61 ◽

Cited By ~ 18

Author(s):

D. Gruffat ◽

S. Gonzalvez ◽

M. Chambard ◽

J. Mauchamp ◽

O. Chabaud

Keyword(s):

Basal Medium ◽

Culture Conditions ◽

Iodide Uptake ◽

Thyroid Cells ◽

Iodide Concentration ◽

Long Term Study ◽

Basal Media ◽

Medium Change ◽

Cells Cultured

ABSTRACT Thyroid cells cultured as monolayers on the porous bottom of culture chambers have been shown to express some specific functions of thyroid follicles. This system, which allows independent access to apical and basal media, is suitable for the long-term study of polarized processes, as the cells maintain their polarized organization. Iodination of thyroglobulin has been investigated under different culture conditions in the presence or absence of TSH. Apical thyroglobulin accumulation, apical iodide concentration and thyroglobulin iodination have been followed simultaneously. Iodide (0·5 μmol/l) was added to basal medium at various stages: only once for 4-day incubations and at each medium change or daily for longer experiments. TSH increased the amount of thyroglobulin secreted into the apical medium by five- to sixfold, whereas high basal iodide concentrations (> 5 μmol/l) inhibited thyroglobulin secretion by TSH-stimulated cells. TSH increased iodide uptake giving an iodide concentration ratio between apical and basal media of about 5. Thyroglobulin iodination was dependent upon TSH. Thyroglobulin was iodinated only in the apical compartment. Secretion and iodination of thyroglobulin were polarized phenomena, but the polarity of iodination was total whereas the polarity of secretion was only partial (10% basal secretion). This functional asymmetry was maintained for up to 29 days. The maximal incorporation of iodine into thyroglobulin obtained was never higher than 3·5 atoms/mol. Apical iodide concentrations from 1 to 15 μmol/l, depending on culture conditions, did not increase this value. These results suggest that cells cultured in this culture system are able to reproduce several steps of thyroidal iodide metabolism although there may be unknown factors which could interfere and reduce the efficiency of thyroglobulin iodination. Journal of Endocrinology (1991) 128, 51–61

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