Mass Generation, Neuron Labeling, and 3D Imaging of Minibrains

Minibrain is a 3D brain in vitro spheroid model, composed of a mixed population of neurons and glial cells, generated from human iPSC derived neural stem cells. Despite the advances in human 3D in vitro models such as aggregates, spheroids and organoids, there is a lack of labeling and imaging methodologies to characterize these models. In this study, we present a step-by-step methodology to generate human minibrain nurseries and novel strategies to subsequently label projection neurons, perform immunohistochemistry and 3D imaging of the minibrains at large multiplexable scales. To visualize projection neurons, we adapt viral transduction and to visualize the organization of cell types we implement immunohistochemistry. To facilitate 3D imaging of minibrains, we present here pipelines and accessories for one step mounting and clearing suitable for confocal microscopy. The pipelines are specifically designed in such a way that the assays can be multiplexed with ease for large-scale screenings using minibrains and other organoid models. Using the pipeline, we present (i) dendrite morphometric properties obtained from 3D neuron morphology reconstructions, (ii) diversity in neuron morphology, and (iii) quantified distribution of progenitors and POU3F2 positive neurons in human minibrains.

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Mass Generation, Neuron Labelling and 3D Imaging of Minibrains

10.1101/2020.07.11.198531 ◽

2020 ◽

Author(s):

Subashika Govindan ◽

Laura Batti ◽

Samira F Osterop ◽

Luc Stoppini ◽

Adrien Roux

Keyword(s):

Large Scale ◽

3D Imaging ◽

Cell Types ◽

Projection Neurons ◽

Neuron Morphology ◽

Mass Generation ◽

One Step ◽

Brain Organoid ◽

Human Ipsc

AbstractMinibrain is a spherical in vitro 3D brain organoid model, composed of a mixed population of neurons and glial cells, generated from human iPSC derived neural stem cells. Despite the advances in human brain organoid models, there is a lack of labelling and imaging methodologies to characterize these models. In this study, we present a step-by-step methodology to generate human minibrain nurseries and novel strategies to subsequently label projection neurons, perform immunohistochemistry and 3D imaging of the minibrains at large multiplexable scales. To visualize projection neurons, we adapt viral transduction and to visualize the organization of cell types we implement immunohistochemistry. To facilitate 3D imaging of minibrains, we present here pipelines and accessories for one step mounting and clearing suitable for confocal microscopy. The pipelines are specifically designed in such a way that the assays can be multiplexed with ease for large-scale screenings using minibrains. Using the pipeline, we present i. dendrite morphometric properties obtained from 3D neuron morphology reconstructions and ii. distribution and quantification of cell types in 3D across whole mount organoids.

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Modulating cell state to enhance suspension expansion of human pluripotent stem cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1714099115 ◽

2018 ◽

Vol 115 (25) ◽

pp. 6369-6374 ◽

Cited By ~ 12

Author(s):

Yonatan Y. Lipsitz ◽

Curtis Woodford ◽

Ting Yin ◽

Jacob H. Hanna ◽

Peter W. Zandstra

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Large Scale ◽

Cell Types ◽

Human Pluripotent Stem Cells ◽

The Body ◽

Altered Expression ◽

Pancreatic Progenitors ◽

Erk Inhibition

The development of cell-based therapies to replace missing or damaged tissues within the body or generate cells with a unique biological activity requires a reliable and accessible source of cells. Human pluripotent stem cells (hPSC) have emerged as a strong candidate cell source capable of extended propagation in vitro and differentiation to clinically relevant cell types. However, the application of hPSC in cell-based therapies requires overcoming yield limitations in large-scale hPSC manufacturing. We explored methods to convert hPSC to alternative states of pluripotency with advantageous bioprocessing properties, identifying a suspension-based small-molecule and cytokine combination that supports increased single-cell survival efficiency, faster growth rates, higher densities, and greater expansion than control hPSC cultures. ERK inhibition was found to be essential for conversion to this altered state, but once converted, ERK inhibition led to a loss of pluripotent phenotype in suspension. The resulting suspension medium formulation enabled hPSC suspension yields 5.7 ± 0.2-fold greater than conventional hPSC in 6 d, for at least five passages. Treated cells remained pluripotent, karyotypically normal, and capable of differentiating into all germ layers. Treated cells could also be integrated into directed differentiated strategies as demonstrated by the generation of pancreatic progenitors (NKX6.1+/PDX1+ cells). Enhanced suspension-yield hPSC displayed higher oxidative metabolism and altered expression of adhesion-related genes. The enhanced bioprocess properties of this alternative pluripotent state provide a strategy to overcome cell manufacturing limitations of hPSC.

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Vertebrate retinal ganglion cells are selected from competent progenitors by the action of Notch

Development ◽

10.1242/dev.121.11.3637 ◽

1995 ◽

Vol 121 (11) ◽

pp. 3637-3650 ◽

Cited By ~ 33

Author(s):

C.P. Austin ◽

D.E. Feldman ◽

J.A. Ida ◽

C.L. Cepko

Keyword(s):

Cell Fate ◽

Ganglion Cells ◽

Notch Pathway ◽

Cell Types ◽

Projection Neurons ◽

Specific Cell ◽

Vitro Assay ◽

Notch Activity

The first cells generated during development of the vertebrate retina are the ganglion cells, the projection neurons of the retina. Although they are one of the most intensively studied cell types within the central nervous system, little is known of the mechanisms that determine ganglion cell fate. We demonstrate that ganglion cells are selected from a large group of competent progenitors that comprise the majority of the early embryonic retina and that differentiation within this group is regulated by Notch. Notch activity in vivo was diminished using antisense oligonucleotides or augmented using a retrovirally transduced constitutively active allele of Notch. The number of ganglion cells produced was inversely related to the level of Notch activity. In addition, the Notch ligand Delta inhibited retinal progenitors from differentiating as ganglion cells to the same degree as did activated Notch in an in vitro assay. These results suggest a conserved strategy for neurogenesis in the retina and describe a versatile in vitro and in vivo system with which to examine the action of the Notch pathway in a specific cell fate decision in a vertebrate.

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Kidney organoid reproducibility across multiple human iPSC lines and diminished off target cells after transplantation revealed by single cell transcriptomics

10.1101/516807 ◽

2019 ◽

Cited By ~ 4

Author(s):

Ayshwarya Subramanian ◽

Eriene-Heidi Sidhom ◽

Maheswarareddy Emani ◽

Nareh Sahakian ◽

Katherine Vernon ◽

...

Keyword(s):

Single Cell ◽

Human Kidney ◽

Cell Types ◽

Mouse Kidney ◽

Target Cells ◽

Rna Seq ◽

Kidney Capsule ◽

Human Ipsc ◽

Kidney Organoids

AbstractHuman iPSC-derived kidney organoids have the potential to revolutionize discovery, but assessing their consistency and reproducibility across iPSC lines, and reducing the generation of off-target cells remain an open challenge. Here, we used single cell RNA-Seq (scRNA-Seq) to profile 415,775 cells to show that organoid composition and development are comparable to human fetal and adult kidneys. Although cell classes were largely reproducible across iPSC lines, time points, protocols, and replicates, cell proportions were variable between different iPSC lines. Off-target cell proportions were the most variable. Prolonged in vitro culture did not alter cell types, but organoid transplantation under the mouse kidney capsule diminished off-target cells. Our work shows how scRNA-seq can help score organoids for reproducibility, faithfulness and quality, that kidney organoids derived from different iPSC lines are comparable surrogates for human kidney, and that transplantation enhances their formation by diminishing off-target cells.

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Biosynthesis And Structural Characterization of Levan By A Recombinant Levansucrase From Bacillus Subtilis ZW019

10.21203/rs.3.rs-511002/v2 ◽

2021 ◽

Author(s):

Jingyue Wang ◽

Xinan Xu ◽

Fangkun Zhao ◽

Nan Yin ◽

Zhijiang Zhou ◽

...

Keyword(s):

Large Scale ◽

Fermentation Broth ◽

Monosaccharide Composition ◽

Enzymatic Reactions ◽

Scale Production ◽

Large Scale Production ◽

Nmr Techniques ◽

One Step ◽

Activity Of Enzymes

Abstract Purpose: The yield of levan extracted from microbial fermentation broth is low, so in vitro catalytic synthesis of levan by levansucrase is expected to be one of the industrial production approaches of levan. Methods: A recombinant plasmid pET-28a-AcmA-Z constructed in the previous study was used to produce levansucrase. The effects of temperature, pH, and metal ions on the levan formation activity of the levansucrase were investigated. The polymer was analyzed by means of HPIC, FTIR, NMR techniques.Results: The recombinant levansucrase could be easily purified in one step and the purified enzyme had a single band clearly visible in SDS-PAGE. The conditions for enzymatic reactions was optimal at pH 5.2 and 40 ℃, and the activity of enzymes was stimulated by K+ and Ca2+. The yield of levan biosynthesis from 10% (w/v) sucrose with 6.45 U/g sucrose of levansucrase was 30.6 g/L. The molecular weight of the levan was about 1.56×106 Da, as measured by GPC. HPIC analysis showed that the monosaccharide composition of the levan was fructose and glucose. The results of FTIR and NMR analysis indicated that the polymer produced by the recombinant levansucrase was β-(2, 6) levan.Conclusions: The results of this study provide a basis for large-scale production of levan by enzymatic method.

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The Subventricular Zone: A Key Player in Human Neocortical Development

The Neuroscientist ◽

10.1177/1073858417691009 ◽

2017 ◽

Vol 24 (2) ◽

pp. 156-170 ◽

Cited By ~ 10

Author(s):

J. Alberto Ortega ◽

Fani Memi ◽

Nevena Radonjic ◽

Radmila Filipovic ◽

Inseyah Bagasrawala ◽

...

Keyword(s):

Cerebral Cortex ◽

Subventricular Zone ◽

Ventricular Zone ◽

Cell Types ◽

Projection Neurons ◽

Cortical Projection ◽

Intrauterine Development ◽

Vitro Model ◽

Primate Cerebral Cortex

One of the main characteristics of the developing brain is that all neurons and the majority of macroglia originate first in the ventricular zone (VZ), next to the lumen of the cerebral ventricles, and later on in a secondary germinal area above the VZ, the subventricular zone (SVZ). The SVZ is a transient compartment mitotically active in humans for several gestational months. It serves as a major source of cortical projection neurons as well as an additional source of glial cells and potentially some interneuron subpopulations. The SVZ is subdivided into the smaller inner (iSVZ) and the expanded outer SVZ (oSVZ). The enlargement of the SVZ and, in particular, the emergence of the oSVZ are evolutionary adaptations that were critical to the expansion and unique cellular composition of the primate cerebral cortex. In this review, we discuss the cell types and organization of the human SVZ during the first half of the 40 weeks of gestation that comprise intrauterine development. We focus on this period as it is when the bulk of neurogenesis in the human cerebral cortex takes place. We consider how the survival and fate of SVZ cells depend on environmental influences, by analyzing the results from in vitro experiments with human cortical progenitor cells. This in vitro model is a powerful tool to better understand human neocortex formation and the etiology of neurodevelopmental disorders, which in turn will facilitate the design of targeted preventive and/or therapeutic strategies.

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Microcarrier Screening and Evaluation for Dynamic Expansion of Human Periosteum-Derived Progenitor Cells in a Xenogeneic Free Medium

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.624890 ◽

2021 ◽

Vol 9 ◽

Author(s):

Kathleen Van Beylen ◽

Ioannis Papantoniou ◽

Jean-Marie Aerts

Keyword(s):

Progenitor Cells ◽

Cell Expansion ◽

Progenitor Cell ◽

Large Scale ◽

Expansion Method ◽

Cell Types ◽

Free Medium ◽

Efficient Expansion

An increasing need toward a more efficient expansion of adherent progenitor cell types arises with the advancements of cell therapy. The use of a dynamic expansion instead of a static planar expansion could be one way to tackle the challenges of expanding adherent cells at a large scale. Microcarriers are often reported as a biomaterial for culturing cells in suspension. However, the type of microcarrier has an effect on the cell expansion. In order to find an efficient expansion process for a specific adherent progenitor cell type, it is important to investigate the effect of the type of microcarrier on the cell expansion. Human periosteum-derived progenitor cells are extensively used in skeletal tissue engineering for the regeneration of bone defects. Therefore, we evaluated the use of different microcarriers on human periosteum-derived progenitor cells. In order to assess the potency, identity and viability of these cells after being cultured in the spinner flasks, this study performed several in vitro and in vivo analyses. The novelty of this work lies in the combination of screening different microcarriers for human periosteum-derived progenitor cells with in vivo assessments of the cells’ potency using the microcarrier that was selected as the most promising one. The results showed that expanding human periosteum-derived progenitor cells in spinner flasks using xeno-free medium and Star-Plus microcarriers, does not affect the potency, identity or viability of the cells. The potency of the cells was assured with an in vivo evaluation, where bone formation was achieved. In summary, this expansion method has the potential to be used for large scale cell expansion with clinical relevance.

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Electrophysiology prediction of single neurons based on their morphology

10.1101/2020.02.04.933697 ◽

2020 ◽

Author(s):

Mario Michiels

Keyword(s):

Data Acquisition ◽

Large Scale ◽

Expert Knowledge ◽

Large Data ◽

Cell Types ◽

Biophysical Model ◽

Single Neurons ◽

Regression Problem ◽

Sophisticated User

AbstractElectrophysiology data acquisition of single neurons represents a key factor for the understanding of neuronal dynamics. However, the traditional method to acquire this data is through patch-clamp technology, which presents serious scalability flaws due to its slowness and complexity to record at fine-grained spatial precision (dendrites and axon).In silico biophysical models are therefore created for simulating hundreds of experiments that would be impractical to recreate in vitro. The optimal way to create these models is based on the knowledge of the morphological and electrical features for each neuron. Since large-scale data acquisition is often unfeasible for electrical data, previous expert knowledge can be used but it must have an acceptable degree of similarity with the type of neurons that we are trying to model.Here, we present a data-driven machine learning approach to predict the electrophysiological features of single neurons in case of only having their morphology available. To solve this multi-output regression problem, we use an artificial neural network that has the particularity of providing a probability distribution for every output feature, to incorporate uncertainty. Input data to train the model is obtained from from the Allen Cell Types database. The electrical properties can depend on the morphology, whose acquisition technology is highly automated and scalable so there exist large data sets of them. We also provide integrations with the BluePyOpt library to create a biophysical model using the original morphology and the predicted electrical features. Finally, we connect the resulting biophysical model with the Geppetto UI software to run all the simulations in a sophisticated user interface.

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An in vivo reporter for tracking lipid droplet dynamics in transparent zebrafish

eLife ◽

10.7554/elife.64744 ◽

2021 ◽

Vol 10 ◽

Author(s):

Dianne Lumaquin ◽

Eleanor Johns ◽

Emily Montal ◽

Joshua M Weiss ◽

David Ola ◽

...

Keyword(s):

Nitric Oxide ◽

Lipid Droplet ◽

Lipid Droplets ◽

Large Scale ◽

Fluorescent Dyes ◽

Cell Types ◽

Structural Protein ◽

Droplet Dynamics

Lipid droplets are lipid storage organelles found in nearly all cell types from adipocytes to cancer cells. Although increasingly implicated in disease, current methods to study lipid droplets in vertebrate models rely on static imaging or the use of fluorescent dyes, limiting investigation of their rapid in vivo dynamics. To address this, we created a lipid droplet transgenic reporter in whole animals and cell culture by fusing tdTOMATO to Perilipin-2 (PLIN2), a lipid droplet structural protein. Expression of this transgene in transparent casper zebrafish enabled in vivo imaging of adipose depots responsive to nutrient deprivation and high-fat diet. Simultaneously, we performed a large-scale in vitro chemical screen of 1280 compounds and identified several novel regulators of lipolysis in adipocytes. Using our Tg(-3.5ubb:plin2-tdTomato) zebrafish line, we validated several of these novel regulators and revealed an unexpected role for nitric oxide in modulating adipocyte lipid droplets. Similarly, we expressed the PLIN2-tdTOMATO transgene in melanoma cells and found that the nitric oxide pathway also regulated lipid droplets in cancer. This model offers a tractable imaging platform to study lipid droplets across cell types and disease contexts using chemical, dietary, or genetic perturbations.

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Biosynthesis and Structural Characterization of Levan by a Recombinant Levansucrase From Bacillus Subtilis ZW019

10.21203/rs.3.rs-511002/v1 ◽

2021 ◽

Author(s):

Jingyue Wang ◽

Xinan Xu ◽

Fangkun Zhao ◽

Nan Yin ◽

Zhijiang Zhou ◽

...

Keyword(s):

Large Scale ◽

Fermentation Broth ◽

Monosaccharide Composition ◽

Enzymatic Reactions ◽

Scale Production ◽

Large Scale Production ◽

Sds Page ◽

One Step ◽

Activity Of Enzymes

Abstract ObjectivesThe yield of levan extracted from microbial fermentation broth is low, so in vitro catalytic synthesis of levan by levansucrase is expected to be one of the industrial production approaches of levan. ResultsA recombinant plasmid Pet-28A-AcmA-Z constructed in the previous study was used to produce levansucrase. The recombinant levansucrase could be easily purified in one step and the purified enzyme had a single band clearly visible in SDS-PAGE. The conditions for enzymatic reactions was optimal at pH 5.2 and 40 ℃, and the activity of enzymes was stimulated by K+ and Ca2+. The yield of levan biosynthesis from 10% (w/v) sucrose with 6.45 U/g sucrose of levansucrase was 30.6 g/L. The molecular weight of the levan was about 1.56×106 Da, as measured by GPC. HPIC analysis showed that the monosaccharide composition of the levan was fructose and glucose. The results of FTIR and NMR analysis indicated that the polymer produced by the recombinant levansucrase was β-(2, 6) levan.ConclusionsThe results of this study provide a basis for large-scale production of levan by enzymatic method.

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