干细胞


分类

现刊
0 Q&A 49 Views Jun 20, 2024

The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME’s intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal bona fide stromal cell population with high precision. The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations. This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche.

往期刊物
0 Q&A 665 Views May 5, 2024

Various protocols have been proven effective in the directed differentiation of mouse and human pluripotent stem cells into skeletal muscles and used to study myogenesis. Current 2D myogenic differentiation protocols can mimic muscle development and its alteration under pathological conditions such as muscular dystrophies. 3D skeletal muscle differentiation approaches can, in addition, model the interaction between the various cell types within the developing organoid. Our protocol ensures the differentiation of human embryonic/induced pluripotent stem cells (hESC/hiPSC) into skeletal muscle organoids (SMO) via cells with paraxial mesoderm and neuromesodermal progenitors’ identity and further production of organized structures of the neural plate margin and the dermomyotome. Continuous culturing omits neural lineage differentiation and promotes fetal myogenesis, including the maturation of fibroadipogenic progenitors and PAX7-positive myogenic progenitors. The PAX7 progenitors resemble the late fetal stages of human development and, based on single-cell transcriptomic profiling, cluster close to adult satellite cells of primary muscles. To overcome the limited availability of muscle biopsies from patients with muscular dystrophy during disease progression, we propose to use the SMO system, which delivers a stable population of skeletal muscle progenitors from patient-specific iPSCs to investigate human myogenesis in healthy and diseased conditions.

0 Q&A 521 Views Apr 5, 2024

Stem cell spheroids are rapidly becoming essential tools for a diverse array of applications ranging from tissue engineering to 3D cell models and fundamental biology. Given the increasing prominence of biotechnology, there is a pressing need to develop more accessible, efficient, and reproducible methods for producing these models. Various techniques such as hanging drop, rotating wall vessel, magnetic levitation, or microfluidics have been employed to generate spheroids. However, none of these methods facilitate the easy and efficient production of a large number of spheroids using a standard 6-well plate. Here, we present a novel method based on pellet culture (utilizing U-shaped
microstructures) using a silicon mold produced through 3D printing, along with a detailed and illustrated manufacturing protocol. This technique enables the rapid production of reproducible and controlled spheroids (for 1 × 106 cells, spheroids = 130 ± 10 μm) from human induced pluripotent stem cells (hIPSCs) within a short time frame (24 h). Importantly, the method allows the production of large quantities (2 × 104 spheroids for 1 × 106 cells) in an accessible and cost-effective manner, thanks to the use of a reusable mold. The protocols outlined herein are easily implementable, and all the necessary files for the method replication are freely available.


Key features

• Provision of 3D mold files (STL) to produce silicone induction device of spheroids using 3D printing.

• Cost-effective, reusable, and autoclavable device capable of generating up to 1.2× 104 spheroids of tunable diameters in a 6-well plate.

• Spheroids induction with multiple hIPSC cell lines.

• Robust and reproducible production method suitable for routine laboratory use.


Graphical overview



Spheroid induction process following the pellet method on molded silicon discs

0 Q&A 667 Views Mar 5, 2024

Here, we describe immunofluorescent (IF) staining assay of 3D cell culture colonoids isolated from mice colon as described previously. Primary cultures developed from isolated colonic stem cells are called colonoids. Immunofluorescence can be used to analyze the distribution of proteins, glycans, and small molecules—both biological and non-biological ones. Four-day-old colonoid cell cultures grown on Lab-Tek 8-well plate are fixed by paraformaldehyde. Fixed colonoids are then subjected to antigen retrieval and blocking followed by incubation with primary antibody. A corresponding secondary antibody tagged with desired fluorescence is used to visualize primary antibody–marked protein. Counter staining to stain actin filaments and nucleus to assess cell structure and DNA in nucleus is performed by choosing the other two contrasting fluorescences. IF staining of colonoids can be utilized to visualize molecular markers of cell behavior. This technique can be used for translation research by isolating colonoids from colitis patients’ colons, monitoring the biomarkers, and customizing their treatments.


Key features

• Analysis of molecular markers of cell behavior.

Protocol to visualize proteins in 3D cell culture.

• This protocol requires colonoids isolated from mice colon grown on matrigel support.

• Protocol requires at least eight days to complete.


Graphical overview


0 Q&A 1468 Views Feb 20, 2024

Astrocytes are increasingly recognized for their important role in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS). In ALS, astrocytes shift from their primary function of providing neuronal homeostatic support towards a reactive and toxic role, which overall contributes to neuronal toxicity and cell death. Currently, our knowledge on these processes is incomplete, and time-efficient and reproducible model systems in a human context are therefore required to understand and therapeutically modulate the toxic astrocytic response for future treatment options. Here, we present an efficient and straightforward protocol to generate human induced pluripotent stem cell (hiPSC)-derived astrocytes implementing a differentiation scheme based on small molecules. Through an initial 25 days, hiPSCs are differentiated into astrocytes, which are matured for 4+ weeks. The hiPSC-derived astrocytes can be cryopreserved at every passage during differentiation and maturation. This provides convenient pauses in the protocol as well as cell banking opportunities, thereby limiting the need to continuously start from hiPSCs. The protocol has already proven valuable in ALS research but can be adapted to any desired research field where astrocytes are of interest.


Key features

• This protocol requires preexisting experience in hiPSC culturing for a successful outcome.

• The protocol relies on a small molecule differentiation scheme and an easy-to-follow methodology, which can be paused at several time points.

• The protocol generates >50 × 106 astrocytes per differentiation, which can be cryopreserved at every passage, ensuring a large-scale experimental output.


Graphical overview


0 Q&A 1126 Views Nov 5, 2023

Brain organoids have been widely used to study diseases and the development of the nervous system. Many reports have investigated the application of brain organoids, but most of these models lack vascular structures, which play essential roles in brain development and neurological diseases. The brain and blood vessels originate from two different germ layers, making it difficult to induce vascularized brain organoids in vitro. We developed this protocol to generate brain-specific blood vessel and cerebral organoids and then fused them at a specific developmental time point. The fused cerebral organoids exhibited robust vascular network-like structures, which allows simulating the in vivo developmental processes of the brain for further applications in various neurological diseases.


Key Features

• Culturing vascularized brain organoids using human embryonic stem cells (hESCs).

• The new approach generates not only neural cells and vessel-like networks but also brain-resident microglia immune cells in a single organoid.


Graphical overview



Workflow and timeline for vessel organoid and vascularized brain organoid generation. (By Figdraw, ID: RTIURffccf)
0 Q&A 664 Views Nov 5, 2023

Induced pluripotent stem cells (iPSCs) generated from human sources are valuable tools for studying skeletal development and diseases, as well as for potential use in regenerative medicine for skeletal tissues such as articular cartilage. To successfully differentiate human iPSCs into functional chondrocytes, it is essential to establish efficient and reproducible strategies that closely mimic the physiological chondrogenic differentiation process. Here, we describe a simple and efficient protocol for differentiation of human iPSCs into chondrocytes via generation of an intermediate population of mesenchymal progenitors. These methodologies include step-by-step procedures for mesenchymal derivation, induction of chondrogenic differentiation, and evaluation of the chondrogenic marker gene expression. In this protocol, we describe the detailed procedure for successful derivation of mesenchymal progenitor population from human iPSCs, which are then differentiated into chondrocytes using high-density culture conditions by stimulating with bone morphogenetic protein-2 (BMP-2) or transforming growth factor beta-3 (TGFβ-3). The differentiated iPSCs exhibit temporal expression of cartilage genes and accumulation of a cartilaginous extracellular matrix in vitro, indicating successful chondrogenic differentiation. These detailed methodologies help effective differentiation of human iPSCs into the chondrogenic lineage to obtain functional chondrocytes, which hold great promise for modeling skeletal development and disease, as well as for potential use in regenerative medicine for cell-based therapy for cartilage regeneration.


Key features

• Differentiation of human iPSCs into chondrocytes using 3D culture methods.

• Uses mesenchymal progenitors as an intermediate for differentiation into chondrocytes.

0 Q&A 494 Views Oct 5, 2023

Adult neural stem/progenitor cells (NSPCs) in two neurogenic areas of the brain, the dentate gyrus and the subventricular zone, are major players in adult neurogenesis. Addressing specific questions regarding NSPCs outside of their niche entails in vitro studies through isolation and culture of these cells. As there is heterogeneity in their morphology, proliferation, and differentiation capacity between these two neurogenic areas, NSPCs should be isolated from each area through specific procedures and media. Identifying region-specific NPSCs provides an accurate pathway for assessing the effects of extrinsic factors and drugs on these cells and investigating the mechanisms of neurogenesis in both healthy and pathologic conditions. A great number of isolation and expansion techniques for NSPCs have been reported. The growth and expansion of NSPCs obtained from the dentate gyrus of aged rats are generally difficult. There are relatively limited data and protocols about NSPCs isolation and their culture from aged rats. Our approach is an efficient and reliable strategy to isolate and expand NSPCs obtained from young adult and aged rats. NSPCs isolated by this method maintain their self-renewal and multipotency.


Key features

• NSPCs isolated from the hippocampal dentate gyrus of young adult and aged rats, based on Kempermann et al. (2014) and Aligholi et al. (2014).

• Maintenance of NSPCs isolated from the dentate gyrus of aged rats (20–24 months) in our culture condition is feasible.

• According to our protocol, maximum growth of primary neurospheres obtained from isolated NSPCs of young and aged rats took 15 and 35 days, respectively.


Graphical overview



Isolation and expansion of neural stem/progenitor cells

0 Q&A 375 Views Sep 5, 2023

Adult stem cells play key roles in homeostasis and tissue repair. These cells are regulated by a tight control of transcriptional programs. For example, muscle stem cells (MuSCs), located beneath the basal lamina, exist in the quiescent state but can transition to an activated, proliferative state upon injury. The control of MuSC state depends on the expression levels of myogenic transcription factors. Recent studies revealed the presence of different mRNA isoforms, with distinct biological regulation. Quantifying the exact expression levels of the mRNA isoforms encoding these myogenic transcription factors is therefore key to understanding how MuSCs switch between cell states. Previously, quantitative real-time polymerase chain reaction (qRT-PCR) has been used to quantify RNA expression levels. However, qRT-PCR depends on large amounts of RNA input and only measures relative abundance. Here, we present a protocol for the absolute quantification of mRNA isoforms using microfluidic digital PCR (mdPCR). Primary MuSCs isolated from individual skeletal muscles (gastrocnemius and masseter) are lysed, and their RNA is reverse-transcribed into cDNA and copied into double-stranded DNA. Following exonuclease I digestion to remove remaining single-stranded DNA, the samples are loaded onto a mdPCR chip with TaqMan probes targeting the mRNA isoforms of interest, whereupon target molecules are amplified in nanoliter chambers. We demonstrate that mdPCR can give exact molecule counts per cell for mRNA isoforms encoding the myogenic transcription factor Pax3. This protocol enables the absolute quantification of low abundant mRNA isoforms in a fast, precise, and reliable way.


Graphical overview



Schematic overview of the workflow. (A) Isolation of individual muscles (gastrocnemius and masseter) from C57/BL6 mice followed by digestion using collagenase II and dispase. (B) Sorting of 500 cells directly into PCR tubes using fluorescence-activated cell sorting (FACS). (C) Reverse transcription of mRNA to cDNA. (D) Polymerase reaction to generate a duplicated cDNA product. (E) Exonuclease I digestion to remove remaining single-stranded DNA and the non-hybridized primers. (F) Denaturation step to inactivate exonuclease I. (G) Loading the samples into the microfluidic chip. (H) Running the TaqMan Digital PCR assay in the Fluidigm Biomark HD real-time PCR machine. (I) Data analysis using the Digital PCR software.

0 Q&A 786 Views Mar 5, 2023

In mammals, the skin comprises several distinct cell populations that are organized into the following layers: epidermis (stratum corneum, stratum granulosum, stratum spinosum, and basal layer), basement membrane, dermis, and hypodermal (subcutaneous fat) layers. It is vital to identify the exact location and function of proteins in different skin layers. Laser capture microdissection (LCM) is an effective technique for obtaining pure cell populations from complex tissue sections for disease-specific genomic and proteomic analysis. In this study, we used LCM to isolate different skin layers, constructed a stratified developmental lineage proteome map of human skin that incorporates spatial protein distribution, and obtained new insights into the role of extracellular matrix (ECM) on stem cell regulation.

0 Q&A 894 Views Nov 20, 2022

Sphingolipids are important structural components of cellular membranes. They also function as prominent signaling molecules to control a variety of cellular events, such as cell growth, differentiation, and apoptosis. Impaired sphingolipid metabolism, particularly defects in sphingolipid degradation, has been associated with many human diseases. Fluorescence sphingolipid analogs have been widely used as efficient probes to study sphingolipid metabolism and intracellular trafficking in living mammalian cells. Compared with nitrobenzoxadiazole fluorophores (NBD FL), the boron dipyrromethene difluoride fluorophores (BODIPY FL) have much higher absorptivity and fluorescence quantum. These features allow more intensive labeling of cells for fluorescence microscopy imaging and flow cytometry analysis. Here, we describe a protocol employing BODIPY FL-labeled sphingolipid analogs to elucidate sphingolipid internalization, trafficking, and endocytosis in mouse embryonic stem cells.


Graphical abstract: