Cell induction Protocols | Pluripotent stem cell | Stem Cell

Alternative Method for Obtaining Human-Induced Pluripotent Stem Cell Lines and Three-Dimensional Growth: A Simplified, Passage-Free Approach that Minimizes Labor

MT Masaya Tsukamoto

TK Tomoyuki Kawasaki

AU Akihiro Umezawa

Hidenori Akutsu

0 Q&A

1983 Views

Oct 5, 2024

Induced pluripotent stem cells (iPSCs) hold significant promise for numerous applications in regenerative medicine, disease modeling, and drug discovery. However, the conventional workflow for iPSC generation, with cells grown under two-dimensional conditions, presents several challenges, including the need for specialized scientific skills such as morphologically assessing and picking colonies and removing differentiated cells during the establishment phase. Furthermore, maintaining established iPSCs in three-dimensional culture systems, while offering scalability, necessitates an enzymatic dissociation step for their further growth in a complex and time-consuming protocol. In this study, we introduce a novel approach to address these challenges by reprogramming somatic cells grown under three-dimensional conditions as spheres using a bioreactor, thereby eliminating the need for two-dimensional culture and colony picking. The iPSCs generated in this study were maintained under three-dimensional conditions simply by transferring spheres to the next bioreactor, without the need for an enzymatic dissociation step. This streamlined method simplifies the workflow, reduces technical variability and labor, and paves the way for future advancements in iPSC research and its wider applications.

Rapid and Simplified Induction of Neural Stem/Progenitor Cells (NSCs/NPCs) and Neurons from Human Induced Pluripotent Stem Cells (hiPSCs)

RK Ryutaro Kajihara

Tadahiro Numakawa

Takumi Era

5 Q&A

7478 Views

Feb 5, 2021

Human induced pluripotent stem cells (iPSCs) and their progeny displaying tissue-specific characteristics have paved the way for regenerative medicine and research in various fields such as the elucidation of the pathological mechanism of diseases and the discovery of drug candidates. iPSC-derived neurons are particularly valuable as it is difficult to analyze neural cells obtained from the central nervous system in humans. For neuronal induction with iPSCs, one of the commonly used approaches is the isolation and expansion of neural rosettes, following the formation of embryonic bodies (EBs). However, this process is laborious, inefficient, and requires further purification of the cells. To overcome these limitations, we have developed an efficient neural induction method that allows for the generation of neural stem/progenitor cells (NSCs/NPCs) from iPSCs within 7 days and of functional mature neurons. Our method yields a PAX6-positive homogeneous cell population, a cortical NSCs/NPCs, and the resultant NSCs/NPCs can be cryopreserved, expanded, and differentiated into functional mature neurons. Moreover, our protocol will be less expensive than other methods since the protocol requires fewer neural supplements during neural induction. This article also presents the FM1-43 imaging assay, which is useful for the presynaptic assessment of the iPSCs-derived human neurons. This protocol provides a quick and simplified way to generate NSCs/NPCs and neurons, enabling researchers to establish in vitro cellular models to study brain disease pathology.

Protocol for Isolation, Stimulation and Functional Profiling of Primary and iPSC-derived Human NK Cells

JM Janine E. Melsen

MT Maria Themeli

Mv Monique M. van Ostaijen-ten Dam

Ev Els van Beelen

GL Gertjan Lugthart

RH Rob C. Hoeben [...]

HM Harald M. Mikkers

+ 1 Author

0 Q&A

7792 Views

Dec 5, 2020

Natural killer (NK) cells are innate immune cells, characterized by their cytotoxic capacity, and chemokine and cytokine secretion upon activation. Human NK cells are identified by CD56 expression. Circulating NK cells can be further subdivided into the CD56^bright(~10%) and CD56^dimNK cell subsets (~90%). NK cell-like cells can also be derived from human induced pluripotent stem cells (iPSC). To study the chemokine and cytokine secretion profile of the distinct heterogenous NK cell subsets, intracellular flow cytometry staining can be performed. However, this assay is challenging when the starting material is limited. Alternatively, NK cell subsets can be enriched, sorted, stimulated, and functionally profiled by measuring secreted effector molecules in the supernatant by Luminex. Here, we provide a rapid and straightforward protocol for the isolation and stimulation of primary NK cells or iPSC-derived NK cell-like cells, and subsequent detection of secreted cytokines and chemokines, which is also applicable for a low number of cells.

Generating Loss-of-function iPSC Lines with Combined CRISPR Indel Formation and Reprogramming from Human Fibroblasts

Andrew M. Tidball

PS Preethi Swaminathan

LD Louis T. Dang

JP Jack M. Parent

1 Q&A

12447 Views

Apr 5, 2018

For both disease and basic science research, loss-of-function (LOF) mutations are vitally important. Herein, we provide a simple stream-lined protocol for generating LOF iPSC lines that circumvents the technical challenges of traditional gene-editing and cloning of established iPSC lines by combining the introduction of the CRISPR vector concurrently with episomal reprogramming plasmids into fibroblasts. Our experiments have produced nearly even numbers of all 3 genotypes in autosomal genes. In addition, we provide a detailed approach for maintaining and genotyping 96-well plates of iPSC clones.

GC/MS-based Analysis of Volatile Metabolic Profile Along in vitro Differentiation of Human Induced Pluripotent Stem Cells

Rosamaria Capuano

Rosa Valentina Talarico

PS Paola Spitalieri

PR Roberto Paolesse

NG Giuseppe Novelli

FS Federica Sangiuolo

Corrado Di Natale

0 Q&A

11369 Views

Dec 5, 2017

Human induced pluripotent stem cells (hiPSCs) are a promising tool in cell-based therapies for degenerative diseases. A safe application of hiPSCs in vivo, requires the detection of the presence of residual undifferentiated pluripotent cells that can potentially cause the insurgence of teratomas. Several studies point out that metabolic products may provide an alternative method to identify the different steps of cells differentiation. In particular, the analysis of volatile organic compounds (VOCs) is gaining a growing interest in this context, thanks to its inherent noninvasiveness. Here, a protocol for VOCs analysis from human induced pluripotent stem cells (hiPSCs) is illustrated. It is based on Solid-Phase Microextraction (SPME) technique coupled with gas chromatography-mass spectrometry (GC/MS). The method is applied to measure the volatile metabolite modifications in cells headspace during cell reprogramming from chorionic villus samples (CVS) to hiPSCs, and along hiPSCs in vitro differentiation into early neural progenitors (NPs), passing through embryoid bodies (EBs) formation.

Exit from Pluripotency Assay of Mouse Embryonic Stem Cells

Daniel Cirera-Salinas

Constance Ciaudo

0 Q&A

9504 Views

Aug 20, 2017

A novel method to assess the dissolution of the core pluripotency transcription-factor circuit of mouse Embryonic Stem Cells (mESCs) has been developed (Ying et al., 2003; Betschinger et al., 2013). In order to efficiently identify genes essential for the break-down of the pluripotency network in mutant mESCs with proliferation defects, we adapted this ‘exit from pluripotency assay’ (Bodak et al., 2017; Cirera-Salinas et al., 2017). The protocol described here has been successfully applied to several mESC lines and is easily transposable from one laboratory to another.

Differentiation of Human Induced Pluripotent Stem Cells (iPS Cells) and Embryonic Stem Cells (ES Cells) into Dendritic Cell (DC) Subsets

SS Stephanie Sontag

MF Malrun Förster

KS Kristin Seré

Martin Zenke

0 Q&A

16638 Views

Aug 5, 2017

Induced pluripotent stem cells (iPS cells) are engineered stem cells, which exhibit properties very similar to embryonic stem cells (ES cells; Takahashi and Yamanaka, 2016). Both iPS cells and ES cells have an extraordinary self-renewal capacity and can differentiate into all cell types of our body, including hematopoietic stem/progenitor cells and dendritic cells (DC) derived thereof. This makes iPS cells particularly well suited for studying molecular mechanisms of diseases, drug discovery and regenerative therapy (Grskovic et al., 2011; Bellin et al., 2012; Robinton and Daley, 2012).

DC are the major antigen presenting cells of the immune system and thus they are key players in modulating and directing immune responses (Merad et al., 2013). DC patrol peripheral and interface tissues (e.g., lung, intestine and skin) to detect invading pathogens, and upon activation they migrate to lymph nodes to activate and prime lymphocytes.

DC comprise a phenotypically heterogeneous family with functionally specialized subsets (Schlitzer and Ginhoux, 2014). Generally, classical DC (cDC) and plasmacytoid DC (pDC) are distinguished, exhibiting a classical and plasma cell-like DC morphology, respectively. cDC recognize a multitude of pathogens and secrete proinflammatory cytokines upon activation, while pDC are specialized to detect intracellular pathogens and secrete type I interferons (Merad et al., 2013; Schlitzer and Ginhoux, 2014). cDC are further divided into cross-presenting cDC1 and conventional cDC2, in the human system referred to as CD141⁺ Clec9a⁺ cDC1 and CD1c⁺ CD14^- cDC2. Human pDC are characterized as CD303⁺ CD304⁺ (Jongbloed et al., 2010; Joffre et al., 2012; Swiecki and Colonna, 2015).

To investigate subset specification and function of human DC, we established a protocol to generate cDC1, cDC2 and pDC in vitro from human iPS cells (or ES cells) (Sontag et al., 2017). Therefore, we differentiated iPS cells (or ES cells), via mesoderm commitment and hemato-endothelial specification, into CD43⁺ CD31⁺ hematopoietic progenitors. Subsequently, those were seeded onto inactivated OP9 stromal cells with FLT3L, SCF, GM-CSF and IL-4 or FLT3L, SCF and GM-CSF to specify cDC1 and cDC2, or cDC1 and pDC, respectively.

Efficient Production of Functional Human NKT Cells from Induced Pluripotent Stem Cells − Reprogramming of Human Vα24⁺iNKT Cells

DY Daisuke Yamada

TI Tomonori Iyoda

KS Kanako Shimizu

YS Yusuke Sato

HK Haruhiko Koseki

SF Shin-ichiro Fujii

0 Q&A

13520 Views

May 20, 2017

Antigen-specific T cell-derived induced pluripotent stem cells (iPSCs) have been shown to re-differentiate into functional T cells and thus provide a potential source of T cells that could be useful for cancer immunotherapy. Human Vα24⁺ invariant natural killer T (Vα24⁺iNKT) cells are subset of T cells that are characterized by the expression of an invariant Vα24-Jα18 paired with Vβ11, that recognize glycolipids, such as α-galactosylceramide (α-GalCer), presented by the MHC class I-like molecule CD1d. Vα24⁺iNKT cells capable of producing IFN-γ are reported to augment anti-tumor responses, which affects both NK cells and CD8⁺ cytotoxic T lymphocytes to eliminate MHC^- and MHC⁺ tumor cells, respectively. Here we describe a robust protocol to reprogram human Vα24⁺iNKT cells into iPSC, and then to re-differentiate them into Vα24⁺iNKT cells (iPS-Vα24⁺iNKT). We further provide a protocol to measure the activity of iPS-Vα24⁺iNKT cells.

Reprogram Murine Epiblast Stem Cells by Epigenetic Inhibitors

HZ Hui Zhang

YD Yali Dou

0 Q&A

7462 Views

Mar 5, 2017

Pluripotent stem cells in the naïve state are highly useful in regenerative medicine and tissue engineering. A robust reprogramming of the primed murine Epiblast Stem Cells (EpiSCs) to naïve pluripotency is feasible via chemical-only approach. This protocol described a method to reprogram murine EpiSCs by MM-401 treatment, which blocks histone H3K4 methylation by MLL1/KMT2A.

iPS Cell Induction from Human Non-T, B cells from Peripheral Blood

KO Keisuke Okita

0 Q&A

9820 Views

Sep 20, 2013

The generation of iPS cells gives an opportunity to use patient-specific somatic cells which are a valuable source for disease modeling and drug discovery. To promote these studies, it is important to make iPS cells from easily accessible and less invasive tissues like blood. Here, we describe the basic method to generate human iPS cells from adult peripheral blood. After the isolation of mononuclear cells, a combination of cytokines stimulates the expansion of hematopoietic stem/progenitor population, which is the main target of this protocol. The cells are transduced with plasmid mixture encoding reprogramming factors. In most cases, the plasmids are lost during the establishment of iPS clones.