Adherent but Not Suspension-Cultured Embryoid Bodies Develop into Laminated Retinal Organoids

Human induced pluripotent stem cells (iPSCs) are differentiated into three-dimensional (3D) retinal organoids to study retinogenesis and diseases that would otherwise be impossible. The complexity and low yield in current protocols remain a technical challenge, particularly for inexperienced personnel. Differentiation protocols require labor-intensive and time-consuming dissection of optic vesicles (OVs). Here we compare this method with a suspension method of developing retinal organoids. iPSCs were differentiated with standard protocols but the suspension-grown method omitted the re-plating of embryoid bodies and dissection of OVs. All other media and treatments were identical between developmental methods. Developmental maturation was evaluated with RT-qPCR and immunocytochemistry. Dissection- and suspension-derived retinal organoids displayed temporal biogenesis of retinal cell types. Differences in retinal organoids generated by the two methods of differentiation included temporal developmental and the organization of neural retina layers. Retinal organoids grown in suspension showed delayed development and disorganized retinal layers compared to the dissected retinal organoids. We found that omitting the re-plating of EBs to form OVs resulted in numerous OVs that were easy to identify and matured along a retinal lineage. While more efficient, the suspension method led to retinal organoids with disorganized retinal layers compared to those obtained using conventional dissection protocols.

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Novel Scalable and Simplified System to Generate Microglia-Containing Cerebral Organoids From Human Induced Pluripotent Stem Cells

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2021.682272 ◽

2021 ◽

Vol 15 ◽

Author(s):

Brittany Bodnar ◽

Yongang Zhang ◽

Jinbiao Liu ◽

Yuan Lin ◽

Peng Wang ◽

...

Keyword(s):

Stem Cells ◽

Human Brain ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Three Dimensional ◽

3D Cell Culture ◽

Cell Types ◽

Embryoid Bodies ◽

Neural Stem Progenitor Cells ◽

Induced Pluripotent

Human cerebral organoid (CO) is a three-dimensional (3D) cell culture system that recapitulates the developing human brain. While CO has proved an invaluable tool for studying neurological disorders in a more clinically relevant matter, there have still been several shortcomings including CO variability and reproducibility as well as lack of or underrepresentation of certain cell types typically found in the brain. As the technology to generate COs has continued to improve, more efficient and streamlined protocols have addressed some of these issues. Here we present a novel scalable and simplified system to generate microglia-containing CO (MCO). We characterize the cell types and dynamic development of MCOs and validate that these MCOs harbor microglia, astrocytes, neurons, and neural stem/progenitor cells, maturing in a manner that reflects human brain development. We introduce a novel technique for the generation of embryoid bodies (EBs) directly from induced pluripotent stem cells (iPSCs) that involves simplified steps of transitioning directly from 3D cultures as well as orbital shaking culture in a standard 6-well culture plate. This allows for the generation of MCOs with an easy-to-use system that is affordable and accessible by any general lab.

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An orthogonal differentiation platform for genomically programming stem cells, organoids, and bioprinted tissues

10.1101/2020.07.11.198671 ◽

2020 ◽

Author(s):

Mark A. Skylar-Scott ◽

Jeremy Y. Huang ◽

Aric Lu ◽

Alex H.M. Ng ◽

Tomoya Duenki ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Pluripotent Stem Cells ◽

Cell Types ◽

Embryoid Bodies ◽

One Pot ◽

Neural Tissues ◽

Induced Pluripotent ◽

And Function ◽

Matrix Free

AbstractSimultaneous differentiation of human induced pluripotent stem cells (hiPSCs) into divergent cell types offers a pathway to achieving tailorable cellular complexity, patterned architecture, and function in engineered human organoids and tissues. Recent transcription factor (TF) overexpression protocols typically produce only one cell type of interest rather than the multitude of cell types and structural organization found in native human tissues. Here, we report an orthogonal differentiation platform for genomically programming stem cells, organoids and bioprinted tissues with controlled composition and organization. To demonstrate this platform, we orthogonally differentiated endothelial cells and neurons from hiPSCs in a one-pot system containing neural stem cell-specifying media. By aggregating inducible-TF and wildtype hiPSCs into pooled and multicore-shell embryoid bodies, we produced vascularized and patterned cortical organoids within days. Using multimaterial 3D bioprinting, we patterned 3D neural tissues from densely cellular, matrix-free stem cell inks that were orthogonally differentiated on demand into distinct layered regions composed of neural stem cells, endothelium, and neurons, respectively. Given the high proliferative capacity and patient-specificity of hiPSCs, our platform provides a facile route for programming cells and multicellular tissues for drug screening and therapeutic applications.

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Preferential Lineage-Specific Differentiation of Osteoblast-Derived Induced Pluripotent Stem Cells into Osteoprogenitors

Stem Cells International ◽

10.1155/2017/1513281 ◽

2017 ◽

Vol 2017 ◽

pp. 1-15 ◽

Cited By ~ 7

Author(s):

Casey L. Roberts ◽

Silvia S. Chen ◽

Angela C. Murchison ◽

Rebecca A. Ogle ◽

Michael P. Francis ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Cell Types ◽

Embryoid Bodies ◽

Population Doubling ◽

Specific Cell ◽

Germ Layer ◽

Induced Pluripotent ◽

Endodermal Cells

While induced pluripotent stem cells (iPSCs) hold great clinical promise, one hurdle that remains is the existence of a parental germ-layer memory in reprogrammed cells leading to preferential differentiation fates. While it is problematic for generating cells vastly different from the reprogrammed cells’ origins, it could be advantageous for the reliable generation of germ-layer specific cell types for future therapeutic use. Here we use human osteoblast-derived iPSCs (hOB-iPSCs) to generate induced osteoprogenitors (iOPs). Osteoblasts were successfully reprogrammed and demonstrated by endogenous upregulation of Oct4, Sox2, Nanog, TRA-1-81, TRA-16-1, SSEA3, and confirmatory hPSC Scorecard Algorithmic Assessment. The hOB-iPSCs formed embryoid bodies with cells of ectoderm and mesoderm but have low capacity to form endodermal cells. Differentiation into osteoprogenitors occurred within only 2–6 days, with a population doubling rate of less than 24 hrs; however, hOB-iPSC derived osteoprogenitors were only able to form osteogenic and chondrogenic cells but not adipogenic cells. Consistent with this, hOB-iOPs were found to have higher methylation of PPARγbut similar levels of methylation on the RUNX2 promoter. These data demonstrate that iPSCs can be generated from human osteoblasts, but variant methylation patterns affect their differentiation capacities. Therefore, epigenetic memory can be exploited for efficient generation of clinically relevant quantities of osteoprogenitor cells.

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Generation of Retinal Organoids with Mature Rods and Cones from Urine-Derived Human Induced Pluripotent Stem Cells

Stem Cells International ◽

10.1155/2018/4968658 ◽

2018 ◽

Vol 2018 ◽

pp. 1-12 ◽

Cited By ~ 6

Author(s):

Guilan Li ◽

Bingbing Xie ◽

Liwen He ◽

Tiancheng Zhou ◽

Guanjie Gao ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Cell Types ◽

Retinal Cell ◽

Retinal Differentiation ◽

Induced Pluripotent ◽

Urine Cells

Urine cells, a body trash, have been successfully reprogrammed into human induced pluripotent stem cells (U-hiPSCs) which hold a huge promise in regenerative medicine. However, it is unknown whether or to what extent U-hiPSCs can generate retinal cells so far. With a modified retinal differentiation protocol without addition of retinoic acid (RA), our study revealed that U-hiPSCs were able to differentiate towards retinal fates and form 3D retinal organoids containing laminated neural retina with all retinal cell types located in proper layer as in vivo. More importantly, U-hiPSCs generated highly mature photoreceptors with all subtypes, even red/green cone-rich photoreceptors. Our data indicated that a supplement of RA to culture medium was not necessary for maturation and specification of U-hiPSC-derived photoreceptors at least in the niche of retinal organoids. The success of retinal differentiation with U-hiPSCs provides many opportunities in cell therapy, disease modeling, and drug screening, especially in personalized medicine of retinal diseases since urine cells can be noninvasively collected from patients and their relatives.

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A novel human pluripotent stem cell-based assay to predict developmental toxicity

Archives of Toxicology ◽

10.1007/s00204-020-02856-6 ◽

2020 ◽

Vol 94 (11) ◽

pp. 3831-3846

Author(s):

Karin Lauschke ◽

Anna Kjerstine Rosenmai ◽

Ina Meiser ◽

Julia Christiane Neubauer ◽

Katharina Schmidt ◽

...

Keyword(s):

Developmental Toxicity ◽

Three Dimensional ◽

Cell Types ◽

Microtiter Plate ◽

Embryoid Bodies ◽

The Body ◽

Chemically Induced ◽

Novel Method ◽

Induced Pluripotent

Abstract There is a great need for novel in vitro methods to predict human developmental toxicity to comply with the 3R principles and to improve human safety. Human-induced pluripotent stem cells (hiPSC) are ideal for the development of such methods, because they are easy to retrieve by conversion of adult somatic cells and can differentiate into most cell types of the body. Advanced three-dimensional (3D) cultures of these cells, so-called embryoid bodies (EBs), moreover mimic the early developing embryo. We took advantage of this to develop a novel human toxicity assay to predict chemically induced developmental toxicity, which we termed the PluriBeat assay. We employed three different hiPSC lines from male and female donors and a robust microtiter plate-based method to produce EBs. We differentiated the cells into cardiomyocytes and introduced a scoring system for a quantitative readout of the assay—cardiomyocyte contractions in the EBs observed on day 7. Finally, we tested the three compounds thalidomide (2.3–36 µM), valproic acid (25–300 µM), and epoxiconazole (1.3–20 µM) on beating and size of the EBs. We were able to detect the human-specific teratogenicity of thalidomide and found the rodent toxicant epoxiconazole as more potent than thalidomide in our assay. We conclude that the PluriBeat assay is a novel method for predicting chemicals’ adverse effects on embryonic development.

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Utilising Induced Pluripotent Stem Cells in Neurodegenerative Disease Research: Focus on Glia

International Journal of Molecular Sciences ◽

10.3390/ijms22094334 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4334

Author(s):

Katrina Albert ◽

Jonna Niskanen ◽

Sara Kälvälä ◽

Šárka Lehtonen

Keyword(s):

Stem Cells ◽

Neurodegenerative Diseases ◽

Neurodegenerative Disease ◽

Induced Pluripotent Stem Cells ◽

Glial Cells ◽

Pluripotent Stem Cells ◽

Cell Types ◽

Human Ipscs ◽

Induced Pluripotent

Induced pluripotent stem cells (iPSCs) are a self-renewable pool of cells derived from an organism’s somatic cells. These can then be programmed to other cell types, including neurons. Use of iPSCs in research has been two-fold as they have been used for human disease modelling as well as for the possibility to generate new therapies. Particularly in complex human diseases, such as neurodegenerative diseases, iPSCs can give advantages over traditional animal models in that they more accurately represent the human genome. Additionally, patient-derived cells can be modified using gene editing technology and further transplanted to the brain. Glial cells have recently become important avenues of research in the field of neurodegenerative diseases, for example, in Alzheimer’s disease and Parkinson’s disease. This review focuses on using glial cells (astrocytes, microglia, and oligodendrocytes) derived from human iPSCs in order to give a better understanding of how these cells contribute to neurodegenerative disease pathology. Using glia iPSCs in in vitro cell culture, cerebral organoids, and intracranial transplantation may give us future insight into both more accurate models and disease-modifying therapies.

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A non-invasive method to generate induced pluripotent stem cells from primate urine

Scientific Reports ◽

10.1038/s41598-021-82883-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Johanna Geuder ◽

Lucas E. Wange ◽

Aleksandar Janjic ◽

Jessica Radmer ◽

Philipp Janssen ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Sendai Virus ◽

Cell Types ◽

Urine Samples ◽

Comparative Approach ◽

Non Invasive ◽

Human Ipscs ◽

Induced Pluripotent

AbstractComparing the molecular and cellular properties among primates is crucial to better understand human evolution and biology. However, it is difficult or ethically impossible to collect matched tissues from many primates, especially during development. An alternative is to model different cell types and their development using induced pluripotent stem cells (iPSCs). These can be generated from many tissue sources, but non-invasive sampling would decisively broaden the spectrum of non-human primates that can be investigated. Here, we report the generation of primate iPSCs from urine samples. We first validate and optimize the procedure using human urine samples and show that suspension- Sendai Virus transduction of reprogramming factors into urinary cells efficiently generates integration-free iPSCs, which maintain their pluripotency under feeder-free culture conditions. We demonstrate that this method is also applicable to gorilla and orangutan urinary cells isolated from a non-sterile zoo floor. We characterize the urinary cells, iPSCs and derived neural progenitor cells using karyotyping, immunohistochemistry, differentiation assays and RNA-sequencing. We show that the urine-derived human iPSCs are indistinguishable from well characterized PBMC-derived human iPSCs and that the gorilla and orangutan iPSCs are well comparable to the human iPSCs. In summary, this study introduces a novel and efficient approach to non-invasively generate iPSCs from primate urine. This will extend the zoo of species available for a comparative approach to molecular and cellular phenotypes.

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Induced Pluripotent Stem Cells (iPSCs) in Vascular Research: from Two- to Three-Dimensional Organoids

Stem Cell Reviews and Reports ◽

10.1007/s12015-021-10149-3 ◽

2021 ◽

Author(s):

Anja Trillhaase ◽

Marlon Maertens ◽

Zouhair Aherrahrou ◽

Jeanette Erdmann

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Induced Pluripotent Stem Cells ◽

Stem Cell Research ◽

Pluripotent Stem Cells ◽

Embryonic Stem ◽

Cell Types ◽

3D Models ◽

Induced Pluripotent

AbstractStem cell technology has been around for almost 30 years and in that time has grown into an enormous field. The stem cell technique progressed from the first successful isolation of mammalian embryonic stem cells (ESCs) in the 1990s, to the production of human induced-pluripotent stem cells (iPSCs) in the early 2000s, to finally culminate in the differentiation of pluripotent cells into highly specialized cell types, such as neurons, endothelial cells (ECs), cardiomyocytes, fibroblasts, and lung and intestinal cells, in the last decades. In recent times, we have attained a new height in stem cell research whereby we can produce 3D organoids derived from stem cells that more accurately mimic the in vivo environment. This review summarizes the development of stem cell research in the context of vascular research ranging from differentiation techniques of ECs and smooth muscle cells (SMCs) to the generation of vascularized 3D organoids. Furthermore, the different techniques are critically reviewed, and future applications of current 3D models are reported. Graphical abstract

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