干细胞

Adipocytes are endocrine cells that function as the main energy storage in our body. They are commonly used in clinical procedures, including their removal via liposuction and transplantation in plastic surgery. Building on this, adipocytes can be used for ex vivo cellular manipulations, enabling therapeutic modifications that can provide beneficial clinical outcomes after transplantation. Here, we provide a detailed protocol on how to modify adipocytes and adipose organoids using CRISPR activation (CRISPRa), a technology termed adipose manipulation transplantation (AMT).

Well-differentiated airway epithelial cultures are commonly used to study airway stem cell lineages, ion and fluid transport, respiratory virus infection and replication, and disease mechanisms in vitro. This culture model involves the isolation and expansion of airway stem cells followed by their differentiation at an air–liquid interface (ALI), a process that has been previously documented in humans and mice. Domestic ferrets (Mustela putorius furo) have gained considerable importance in respiratory disease research due to their notable susceptibility to these conditions and their anatomical similarities to humans. Here, we present a comprehensive description of the isolation and culture of stem/progenitor cells from the ferret airway, along with a protocol for their differentiation at the ALI. Our findings have demonstrated that this ferret culture system not only supports the differentiation of the predominant airway epithelial cell types but also facilitates the generation of rare airway epithelial subpopulations, including pulmonary ionocytes, tuft cells, and pulmonary neuroendocrine cells. Additionally, we provide a detailed procedure for measuring transepithelial ion transport relevant to airway diseases, particularly cystic fibrosis. The ability to isolate and culture ferret airway stem cells, combined with ALI differentiation and functional assessment of transepithelial ion transport, offers a powerful platform for evaluating genetic and pharmacologic interventions related to cystic fibrosis.

The fatal motor neuron (MN) disease amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of the phrenic MNs (phMNs) controlling the activity of the diaphragm, leading to death by respiratory failure. Human experimental models to study phMNs are lacking, hindering the understanding of the mechanisms of phMN degeneration in ALS. Here, we describe a protocol to derive phrenic-like MNs from human induced pluripotent stem cells (hiPSC-phMNs) within 30 days. During spinal cord development, phMNs emerge from specific MN progenitors located in the dorsalmost MN progenitor (pMN) domain at cervical levels, under the control of a ventral-to-dorsal gradient of Sonic hedgehog (SHH) signaling and a rostro-caudal gradient of retinoic acid (RA). The method presented here uses optimized concentrations of RA and the SHH agonist purmorphamine, followed by fluorescence-activated cell sorting (FACS) of the resulting MN progenitor cells (MNPCs) based on a cell-surface protein (IGDCC3) enriched in hiPSC-phMNs. The resulting cultures are highly enriched in MNs expressing typical phMN markers. This protocol enables the generation of hiPSC-phMNs and is highly reproducible using several hiPSC lines, offering a disease-relevant system to study mechanisms of respiratory MN dysfunction. While the protocol has been validated in the context of ALS research, it can be adopted to study human phrenic MNs in other research fields where these neurons are of interest.

Cancer-associated mesenchymal stem cells (Ca-MSCs), an integral part of the tumor microenvironment, play a major role in modulating tumor progression; they have been reported to progress as well as inhibit various cancers, including cervical cancer. To understand the exact role of Ca-MSCs in tumor modulation, it is necessary to have an optimized protocol for Ca-MSCs isolation. This work demonstrates the isolation and expansion of a primary culture of cervical cancer–associated MSCs (CCa-MSCs) from the biopsy sample of cervical cancer patients using the explant culture technique. The isolated cells were characterized according to International Society for Cellular Therapy (ISCT) guidelines. Morphological analysis revealed that cells were adherent to the plastic surface and possessed spindle-shaped morphology. Flow cytometry analysis of the cells showed high expression (~98%) for MSC-specific cell surface markers (CD90, CD73, and CD105), negative expression (<0.5%) for endothelial cell marker (CD34) and hematopoietic cell marker (CD45), and negligible expression for HLA-DR, as recommended by ISCT. Further, trilineage differentiation potential analysis of the cells showed their osteogenic and chondrogenic potential and adipogenic differentiation. This standardized protocol will assist in the cultivation of CCa-MSCs and the study of their interactions with tumor cells and other components of the tumor microenvironment. This protocol may be utilized in the establishment of Ca-MSCs from other types of cancers as well.

Skeletal muscle–specific stem cells are responsible for regenerating damaged muscle tissue following strenuous physical activity. These muscle stem cells, also known as satellite cells (SCs), can activate, proliferate, and differentiate to form new skeletal muscle cells. SCs can be identified and visualized utilizing optical and electron microscopy techniques. However, studies identifying SCs using fluorescent imaging techniques vary significantly within their methodology and lack fundamental aspects of the guidelines for rigor and reproducibility that must be included within immunohistochemical studies. Therefore, a standardized method for identifying human skeletal muscle stem cells is warranted, which will improve the reproducibility of future studies investigating satellite activity. Additionally, although it has been suggested that SC shape can change after exercise, there are currently no methods for examining SC morphology. Thus, we present an integrated workflow for three-dimensional visualization of satellite cell nuclei, validated by the spatial context of the fluorescent labeling and multichannel signal overlap. Our protocol includes, from start to finish, post-biopsy extraction and embedding, tissue sectioning, immunofluorescence, imaging steps and acquisition, and three-dimensional data post-processing. Because of the depth volume generated from the confocal microscope z-stacks, this will allow future studies to investigate the morphology of SC nuclei and their activity, instead of traditionally observing them in two-dimensional space (x, y).

Bone repair is a complex regenerative process relying on skeletal stem/progenitor cells (SSPCs) recruited predominantly from the periosteum. Activation and differentiation of periosteal SSPCs occur in a heterogeneous environment, raising the need for single cell/nucleus transcriptomics to decipher the response of the periosteum to injury. Enzymatic cell dissociation can induce a stress response affecting the transcriptome and lead to overrepresentation of certain cell types (i.e., immune and endothelial cells) and low coverage of other cell types of interest. To counteract these limitations, we optimized a protocol to isolate nuclei directly from the intact periosteum and from the fracture callus to perform single-nucleus RNA sequencing. This protocol is adapted for fresh murine periosteum, fracture callus, and frozen human periosteum. Nuclei are isolated using mechanical extraction combined with fluorescence-based nuclei sorting to obtain high-quality nucleus suspensions. This protocol allows the capture of the full diversity of cell types in the periosteum and fracture environment to better reflect the in vivo tissue composition.

Primary human intestinal stem cells (ISCs) can be cultured and passaged indefinitely as two-dimensional monolayers grown on soft collagen. Culturing ISCs as monolayers enables easy access to the luminal side for chemical treatments and provides a simpler topology for high-resolution imaging compared to cells cultured as three-dimensional organoids. However, the soft collagen required to support primary ISC growth can pose a challenge for live imaging with an inverted microscope, as the collagen creates a steep meniscus when poured into wells. This may lead to uneven growth toward the center of the well, with cells at the edges often extending beyond the working distance of confocal microscopes. We have engineered a 3D-printed collagen mold that enables the preparation of chamber slides with flat, smooth, and reproducible thin collagen layers. These layers are adequate to support ISC growth while being thin enough to optimize live imaging with an inverted microscope. We present methods for constructing the collagen press, preparing chamber slides with pressed collagen, and plating primary human ISCs for growth and analysis.

Corneal scarring, a significant cause of global blindness, results from various insults, including trauma, infections, and genetic disorders. The conventional treatment to replace scarred corneal tissues includes partial or full-thickness corneal transplantation using healthy donor corneas. However, only 1 in 70 individuals with treatable corneal scarring can undergo surgery, due to the limited supply of transplantable donor tissue. Our research focuses on cell-based strategies, specifically ex vivo–expanded corneal stromal stem cells (CSSCs), to address corneal scarring. Preclinical studies have demonstrated the efficacy of CSSC treatment in reducing corneal inflammation and fibrosis, inhibiting scar formation, and regenerating native stromal tissue. Mechanisms include CSSC differentiation into stromal keratocytes and the expression of regenerative cytokines. Here, we present a good manufacturing practice (GMP)-compliant protocol to isolate and expand human CSSCs. This method paves the way to produce clinical-grade CSSCs for transplantation and clinical trials.

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.

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