Stem Cell

Planarians are free-living flatworms that emerged as a crucial model system to understand regeneration and stem cell biology. The ability to purify neoblasts, the adult stem cell population of planaria, through fluorescence-activated cell sorting (FACS) has tremendously increased our understanding of pluripotency, specialization, and heterogeneity. To date, the FACS-based purification methods for neoblasts relied on nuclear dyes that discriminate proliferating cells (>2N), as neoblasts are the only dividing somatic cells. However, this method does not distinguish the functional states within the neoblast population. Our work has shown that among the neoblasts, the pluripotent stem cells (PSCs) are associated with low mitochondrial content and this property could be leveraged for purification of the PSC-enriched population. Using the mitochondrial dye MitoTracker Green (MTG) and the nuclear dye SiR-DNA, we have described a method for isolation of PSCs that are viable and compatible with downstream experiments, such as transplantation and cell culture. In this protocol, we provide a detailed description for sample preparation and FACS gating for neoblast isolation in planaria.

MicroRNAs are small RNAs that negatively regulate gene expression and play an important role in fine-tuning molecular pathways during development. There is increasing interest in studying their function in the kidney, but the majority of studies to date use kidney cell lines and assess the total amounts of miRNAs of interest either by qPCR or by high-throughput methods such as next generation sequencing. However, this provides little information as to the distribution of the miRNAs in the developing kidney, which is crucial in deciphering their role, especially as there are multiple kidney cell types, each with its own specific transcriptome. Thus, we present a protocol for obtaining spatial information for miRNA expression during kidney development by in situ hybridization (ISH) of anti-miRNA, digoxigenin-labelled (DIG), Locked Nucleic Acid (LNA^®) probes on (i) native human embryonic tissue and (ii) human pluripotent stem cell (hPSC)-derived 3D kidney organoids that model kidney development. We found that the method reveals the precise localization of miRNA in specific anatomical structures and/or cell types and confirms their absence from others, thus informing as to their specific role during development.

Odor-detecting olfactory sensory neurons residing in the nasal olfactory epithelium (OE) are the only neurons in direct contact with the external environment. As a result, these neurons are subjected to chemical, physical, and infectious insults, which may be the underlying reason why neurogenesis occurs in the OE of adult mammals. This feature makes the OE a useful model for studying neurogenesis and neuronal differentiation, with the possibility for systemic as well as local administration of various compounds and infectious agents that may interfere with these cellular processes. Several different chemical compounds have been shown to cause toxic injury to the OE, which can be used for OE ablation. We, and others, have found that the systemic administration of the hyperthyroid drug, methimazole, reliably causes olfactotoxicity as a side effect. Here, we outline an OE lesioning protocol for single or repeated ablation by methimazole. A single methimazole administration can be used to study neuroepithelial regeneration and stem cell activation, while repeated ablation and regeneration of OE enable the study of tissue stem cell exhaustion and generation of tissue metaplasia.

The skin is the largest organ that protects our body from the external environment and it is constantly exposed to pathogenic insults and injury. Repair of damage to this organ is carried out by a complex process involving three overlapping phases of inflammation, proliferation and remodeling. Histological analysis of wounded skin is a convenient approach to examine broad alterations in tissue architecture and investigate cells in their indigenous microenvironment. In this article we present a protocol for immunohistochemical examination of wounded skin to study mechanisms involved in regulating stem cell activity, which is a vital component in the repair of the damaged tissue. Performing such histological analysis enables the understanding of the spatial relationship between cells that interact in the specialized wound microenvironment. The analytical tools described herein permit the quantitative measurement of the regenerative ability of stem cells adjacent to the wound and the extent of re-epithelialization during wound closure. These protocols can be adapted to investigate numerous cellular processes and cell types within the wounded skin.

Pluripotent stem cells (PSCs) have the potential to provide homogeneous cell populations of T cells that can be grown at a clinical scale and genetically engineered to meet specific clinical needs. OP9-DLL4, a stromal line ectopically expressing the Notch ligand Delta-like 4 (DLL4) is used to support differentiation of PSCs to T-lymphocytes. This article outlines several protocols related to generation of T cells from human and non-human primate (NHP) PSCs, including initial hematopoietic differentiation of PSC on OP9 feeders or defined conditions, followed by coculture of the OP9-DLL4 cells with the PSC-derived hematopoietic progenitors (HPs), leading to efficient differentiation to T lymphocytes. In addition, we describe a protocol for robust T cell generation from hPSCs conditionally expressing ETS1. The presented protocols provide a platform for T cell production for disease modeling and evaluating their use for immunotherapy in large animal models.

For years, the mammary gland serves as a perfect example to study the self-renew and differentiation of adult stem cells, and the regulatory mechanisms of these processes as well. To assess the function of given genes and/or other factors on stemness of mammary cells, several In vitro assays were developed, such as mammospheres formation assay, detection of stem cell markers by mRNA expression or flow cytometry and so on. However, the capacity of reconstruction of whole mount in the cleared fat pad of recipient female mice is a golden standard to estimate the stemness of the cells. Here we described a step-by-step protocol for in vivo mammary gland formation assay, including preparation of “cleared” recipients and mammary cells for implantation, the surgery process and how to assess the experimental results. Combined with manipulation of mammary cells via gene editing and /or drug treatment, this protocol could be very useful in the researches of mammary stem cells and mammary development.

Unlike mammals, primitive vertebrates have immense capability to regenerate almost all of their organs including the central nervous system. Among primitive organisms, zebrafish have been extensively used as a model system for regeneration studies. The retina is a part of the central nervous system and mammals lack the potential to repair any damage caused to it. Zebrafish have been used for retina regeneration studies because of ease in handling and maintenance. In zebrafish, Muller glia cells respond to damage and enter the regenerative cascade to maintain the retinal homeostasis. Zebrafish retinal damage can be induced by light, chemical or mechanical methods. Here we are describing the mechanical method of retinal injury, which ensures uniform damage to all retinal layers. Alongside this, we have also described in vivo manipulation strategies for the regeneration associated genes and preparation of retinal tissue for immunohistochemical analysis.

Intra-embryo genome editing by CRISPR/Cas9 has enabled rapid generation of gene knockout animals. However, large fragment knock-in directly into embryos’ genome is still difficult, especially without microinjection of donor DNA. Viral vectors are good transporters of knock-in donor DNA for cell lines, but seemed unsuitable for pre-implantation embryos with zona pellucida, glycoprotein membrane surrounding early embryos. We found adeno-associated virus (AAV) can infect zygotes of various mammals through intact zona pellucida. AAV-mediated donor DNA delivery following Cas9 ribonucleoprotein electroporation enables large fragment knock-in without micromanipulation.