Current Protocols in Stem Cell Biology

Monocytes and macrophages are essential for immune defense and tissue hemostasis. They are also the underlying trigger of many diseases. The availability of robust and short protocols to induce monocytes and macrophages from human induced pluripotent stem cells (hiPSCs) will benefit many applications of immune cells in biomedical research. Here, we describe a protocol to derive and functionally characterize these cells. Large numbers of hiPSC-derived monocytes (hiPSC-mono) could be generated in just 15 days...

Pluripotent stem cell (PSC) cultures are subjected to selective pressures that can result in acquisition and expansion of recurrent genetic abnormalities at any time. These recurrent abnormalities enhance the variant cells harboring them with a competitive advantage over wild-type cells. Variant cells can eventually supplant wild-type cells entirely and become fixed in culture. Such variants can impact the efficacy of PSCs in research and clinical applications. Therefore, routine genomic characterization is required for reliable and effective use of PSCs...

The homeostatic proliferation-differentiation gradient in the esophageal epithelium is perturbed under inflammatory disease conditions such as gastroesophageal reflux disease and eosinophilic esophagitis. Herein we describe the protocols for rapid generation (<14 days) and characterization of single-cell-derived, three-dimensional (3D) esophageal organoids from human subjects and mice with normal esophageal mucosa or inflammatory disease conditions. While 3D organoids recapitulate normal epithelial renewal, proliferation, and differentiation, non-cell autonomous reactive epithelial changes under inflammatory conditions are evaluated in the absence of the inflammatory milieu...

Genome editing has become one of the most powerful tools in present-day stem cell and regenerative medicine research, but despite its rapid acceptance and widespread use, some elements of the technology still need improvement. In this unit, we present data regarding the use of a new, more efficient type of transcription activator-like effector nuclease (TALEN) for gene editing. Our group has generated bicistronic genes in which classical TALEN coding sequences are linked by 2A elements to different reporter molecules, such as fluorochromes (TALEN-F) or membrane receptors (TALEN-M)...

Translating human induced pluripotent stem cell (hiPSC)-derived cells and tissues into the clinic requires streamlined and reliable production of clinical-grade hiPSCs. This article describes an entirely animal component-free procedure for the reliable derivation of stable hiPSC lines from donor peripheral blood mononuclear cells (PBMCs) using only autologous patient materials and xeno-free reagents. PBMCs are isolated from a whole blood donation, from which a small amount of patient serum is also generated...

The complex program of mouse development entails specification of the embryonic epiblast (Epi) as well as the extra-embryonic trophectoderm (TE) and primitive endoderm (PrE). These three lineages of mouse blastocyst can be modeled in vitro using stem cells derived from primary tissues. In these cultures, cells self-renew while retaining their developmental potential if put back into a developing embryo. Indeed, embryonic stem cells (ESC), when injected into a blastocyst, readily contribute to all embryonic lineages...

TP53 point mutations are found in 50% of all cancers and seem to play an important role in cancer pathogenesis. Thus, human induced pluripotent stem cells (hiPSCs) overexpressing mutant TP53 are a valuable tool for the generation of in vitro models of cancer stem cells or for in vivo xenograft models. Here, we describe a protocol for the alteration of gene expression in hiPSCs via overexpression of a mutant form of the TP53 (R249S) gene using lentiviral transduction. A high amount of TP53 protein is detected 1 week after transduction and antibiotic selection...

Skeletal muscle tissue regeneration requires quiescent satellite cell activation, proliferation, and differentiation. Regenerative capacity of satellite cells can be studied in vitro by differentiating under low-serum conditions (2% to 5%) to form multinucleated myotubes. Myotubes are fixed and stained, and indices of differentiation are quantified. Jenner and Giemsa stains are typically used for myotube staining; however, this staining process can be variable depending on factors such as stain pH, staining time, and time since stain preparation...

In the 20 years since the first human pluripotent stem cell (hPSC) lines were established, there have been a plethora of protocols developed that allow us to generate a wide range of human cell types in vitro. Efforts to achieve a greater degree of specificity and efficiency in generating desired cell types have resulted in increasingly complex approaches. The magnitude and timing of signals has become key, and the concept of a "fully defined" system is a forever sought-after goal with shifting goalposts...

Morphogens are biological molecules that alter cellular identity and behavior across both space and time. During embryonic development, morphogen spatial localization can be confined to small volumes in a single tissue or permeate throughout an entire organism, and the temporal effects of morphogens can range from fractions of a second to several days. In most cases, morphogens are presented as a gradient to adjacent cells within tissues to pattern cell fate. As such, to appropriately model development and build representative multicellular architectures in vitro, it is vital to recapitulate these gradients during stem cell differentiation...

Mutation of the gene GJB2, encoding connexin 26 (CX26; also known as gap junction beta 2), is the most frequent cause of hereditary deafness worldwide. CX26 is expressed in cochlear nonsensory cells, such as cochlear supporting cells, and forms gap junction plaques (GJPs) at cell-cell borders. Cochlear CX26-GJP-forming cells (Cx26GJCs) are thought to be an important therapeutic target for treatment of hereditary deafness. Nevertheless, the generation of Cx26GJCs-such as cochlear supporting cells-from embryonic stem/induced pluripotent stem (ES/iPS) cells has not been reported to date...

Our laboratory and others have developed protocols to generate glucose-responsive stem cell-derived β cells in vitro. The cells resulting from these protocols could supplement or replace the use of human cadaveric islets for cell-based therapy for diabetes. The combination of an unlimited supply of pluripotent stem cell-derived β cells and gene-editing approaches will facilitate numerous in vitro studies not possible with cadaveric islets. Here, we describe a protocol for fluorescent labeling and isolation of stem cell-derived β cells...

Human pluripotent stem cell (hPSC) derivatives are valuable for a variety of research applications and have the potential to revolutionize approaches to personalized medicine. However, differentiation efficiency varies among cell lines and protocols. Therefore, methods to reliably determine cell type identity in cultures of hPSC derivatives in a manner that is consistent among laboratories are needed. While flow cytometry is apt for routine assessment of population heterogeneity, standardized protocols are not available for most cell types...

This unit describes a protocol for generating retinal organoids that contain all major retinal cell types and are responsive to light from human pluripotent stem cells (hPSCs). hPSCs are differentiated in 96-well plates to allow large-scale production of organoids that could be used for multiple applications, including study of human retinal development, disease modeling, and compound screening. The differentiation approach is based on the knowledge that insulin-like growth factor 1 signaling together with retinoic acid and triiodothyronine is important for retinal development...

We previously established a two-step protocol for differentiation of human pluripotent stem cells (hPSCs) into trophoblasts, using a StemPro-based minimal medium (EMIM) with bone morphogenetic protein-4 (BMP4). This protocol was suboptimal, resulting in induction of mixed mesoderm and trophoblast markers. Furthermore, adapting hPSCs to StemPro has proven difficult, and prolonged culture in this medium has been shown to promote genomic instability. Therefore, we moved on to the use of new media, including E8, and most recently, StemFlex, for rapid adaptation from feeder to non-feeder conditions...

Sympathetic neurons are crucial for maintenance of body homeostasis and regulation of all organs. Diseases can arise from malfunction of sympathetic neurons, including malignancies, hypertension, and genetic disorders. Human pluripotent stem cells (hPSCs) allow modeling of human diseases and the in-depth study of pathologies of specific cell types associated with such disorders. Advances in the ability to differentiate hPSCs in vitro has allowed the generation of specific cell types such as sympathetic neurons, which provides the novel opportunity to study diseases affecting the sympathetic nervous system in the human context...

This article describes a screening platform to identify compounds that affect human embryonic vascular development. The procedure comprises the generation of human embryonic-like endothelial cells (ECs) from human pluripotent stem cells (hPSCs) and subsequent maturation under arterial flow conditions; the use of these cells for the high-throughput screening of small molecules that specifically inhibit the survival of embryonic-like ECs; the confirmation of the hits in embryonic-like ECs cultured under flow shear stress; and final validation in mouse embryonic ECs...

Pluripotency refers to the capacity of single cells to form derivatives of the three germ layers-ectoderm, mesoderm, and endoderm. Pluripotency can be captured in vitro as a spectrum of pluripotent stem cell states stabilized in specialized laboratory conditions. The recent discovery that pluripotent stem cells can colonize the embryos of distantly related animal organisms could, with further refinement, enable the generation of chimeric embryos composed of cells of human and animal origin. If achievable, the production of human-animal chimeras will open up new opportunities for regenerative medicine, facilitating human disease modeling and human organ generation inside large animals...

Embryonic stem cells are pluripotent whereas adult stem cells are multipotent in nature. In recent years, evidence suggests that adult stem cells not only differentiate into specific cell lineages but also transdifferentiate into multiple cell lineages. Progenitor cells are found in adult bone marrow, blood, and other organs and differentiate into numerous cell lineages regardless of origin. Identifying a subset that can differentiate into mature endothelial cells is essential. This article describes peripheral blood collection in adult beagle dogs, isolation of peripheral blood mononuclear cells (PBMNCs) from the cell fraction, separation of a subset of CD34+ cells using immunomagnetic principles, characterization of PBMNCs and CD34+ cells using flow cytometry, and evaluation of gene expression of CD34, KDR, and CD133 in CD34+ fractions...

Human induced pluripotent stem cells (hiPSCs) not only offer great opportunities for the study of human development but also have tremendous potential for future clinical cell-based therapies. The protocol outlined here is used to differentiate hiPSCs into lung epithelial cell types through a process that faithfully recapitulates the stepwise events observed in vivo. From pluripotency, cells are differentiated to a definitive endoderm fate, followed by progression into anteriorized foregut endoderm that has the ability to give rise to both proximal and distal epithelial cells...