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Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.

The derivation of human embryonic stem cells followed by the discovery of induced pluripotent stem cells and leaps in genome editing approaches have continuously fueled enthusiasm for the development of new models of neurodegenerative diseases such as Parkinson’s disease (PD). PD is characterized by the relative selective loss of dopaminergic neurons (DNs) in specific areas of substantia nigra pars compacta (SNpc). While degeneration in late stages can be widespread, there is stereotypic early degeneration of these uniquely vulnerable neurons. Various causes of selective vulnerability have been investigated but much remains unclear. Most studies have sought to identify cell autonomous properties of the most vulnerable neurons. However, recent findings from genetic studies and model systems have added to our understanding of non-cell autonomous contributions including regional-specific neuro-immune interactions with astrocytes, resident or damage-activated microglia, neuro-glia cell metabolic interactions, involvement of endothelial cells, and damage to the vascular system. All of these contribute to specific vulnerability and, along with aging and environmental factors, might be integrated in a complex stressor-threshold model of neurodegeneration. In this forward-looking review, we synthesize recent advances in the field of PD modeling using human pluripotent stem cells, with an emphasis on organoid and complex co-culture models of the nigrostriatal niche, with emerging CRISPR applications to edit or perturb expression of causal PD genes and associated risk factors, such asGBA, to understand the impact of these genes on relevant phenotypes.

Summary Here, we describe a high-throughput 3D differentiation protocol for deriving midbrain dopaminergic neurons from human pluripotent stem cells. The use of organoids has become prevalent in disease modeling, but there is a high demand for more homogeneous cultures. Our approach is advantageous for large-scale production of uniform midbrain organoids that can be maintained in diverse formats, and our reporters allow for sorting of dopaminergic neurons. The maturing long-term organoid cultures can be used as a model for the entire midbrain. For complete details on the use and execution of this protocol, please refer to Ahfeldt et al. (2020)., Graphical abstract, Highlights • High-throughput generation of 3D midbrain organoids from hPSCs in spinner flasks • Generation of reporter hPSC lines for dopaminergic neurons • Dopaminergic neurons cultured in 2D, 2.5D, and 3D formats • Maturation in long-term midbrain organoid cultures, Here, we describe a high-throughput 3D differentiation protocol for deriving midbrain dopaminergic neurons from human pluripotent stem cells. The use of organoids has become prevalent in disease modeling, but there is a high demand for more homogeneous cultures. Our approach is advantageous for large-scale production of uniform midbrain organoids that can be maintained in diverse formats, and our reporters allow for sorting of dopaminergic neurons. The maturing long-term organoid cultures can be used as a model for the entire midbrain.

Utilizing patient derived cells has enormous promise for discovering new drugs for diseases of the nervous system, a goal that has been historically quite challenging. In this review, we will outline the potential of human stem cell derived neuron models for assessing therapeutics and high-throughput screening and compare to more traditional drug discovery strategies. We summarize recent successes of the approach and discuss special considerations for developing human stem cell based assays. New technologies, such as genome editing, offer improvements to help overcome the challenges that remain. Finally, human neurons derived from patient cells have advantages for translational research beyond drug screening as they can also be used to identify individual efficacy and safety prior to clinical testing and for dissecting disease mechanisms. This article is part of a Special Issue entitled SI: Exploiting human neurons.

Bipolar disorder (BD) is a common neuropsychiatric disorder characterized by chronic recurrent episodes of depression and mania. Despite evidence for high heritability of BD, little is known about its underlying pathophysiology. To develop new tools for investigating the molecular and cellular basis of BD we applied a family-based paradigm to derive and characterize a set of 12 induced pluripotent stem cell (iPSC) lines from a quartet consisting of two BD-affected brothers and their two unaffected parents. Initially, no significant phenotypic differences were observed between iPSCs derived from the different family members. However, upon directed neural differentiation we observed that CXCR4 (CXC chemokine receptor-4) expressing central nervous system (CNS) neural progenitor cells (NPCs) from both BD patients compared to their unaffected parents exhibited multiple phenotypic differences at the level of neurogenesis and expression of genes critical for neuroplasticity, including WNT pathway components and ion channel subunits. Treatment of the CXCR4+ NPCs with a pharmacological inhibitor of glycogen synthase kinase 3 (GSK3), a known regulator of WNT signaling, was found to rescue a progenitor proliferation deficit in the BD-patient NPCs. Taken together, these studies provide new cellular tools for dissecting the pathophysiology of BD and evidence for dysregulation of key pathways involved in neurodevelopment and neuroplasticity. Future generation of additional iPSCs following a family-based paradigm for modeling complex neuropsychiatric disorders in conjunction with in-depth phenotyping holds promise for providing insights into the pathophysiological substrates of BD and is likely to inform the development of targeted therapeutics for its treatment and ideally prevention.

The utility of human pluripotent stem cells is dependent on efficient differentiation protocols that convert these cells into relevant adult cell types. Here we report the robust and efficient differentiation of human pluripotent stem cells into white or brown adipocytes. We found that inducible expression of PPARG2 alone or combined with CEBPB and/or PRDM16 in mesenchymal progenitor cells derived from pluripotent stem cells programmed their development towards a white or brown adipocyte cell fate with efficiencies of 85%-90%. These adipocytes retained their identity independent of transgene expression, could be maintained in culture for several weeks, expressed mature markers and had mature functional properties such as lipid catabolism and insulin-responsiveness. When transplanted into mice, the programmed cells gave rise to ectopic fat pads with the morphological and functional characteristics of white or brown adipose tissue. These results indicate that the cells could be used to faithfully model human disease.

The reprogramming of somatic cells into induced pluripotent stem (iPS) cells upon overexpression of the transcription factors Oct4, Sox2, Klf4 and cMyc is extremely inefficient. It has been assumed that the somatic differentiation state provides a barrier for efficient reprogramming; however, direct evidence for this notion is lacking. Here, we have tested the susceptibilities of hematopoietic cells at different stages of differentiation to be reprogrammed into iPS cells. Surprisingly, hematopoietic stem and progenitor cells give rise to iPS cells up to 300 times more efficiently compared with terminally differentiated B and T cells, yielding reprogramming efficiencies of up to 28%. Our data provide evidence that the differentiation stage of the starting cell has a critical influence on the efficiency of reprogramming into iPS cells. Moreover, we identify adult hematopoietic progenitors as an attractive cell type for applications of iPS technology in research and therapy.

Summary: Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report here the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells, the latter for which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease—dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor-neuron death, and hepatitis C infection. We found little evidence of TALEN off-target effects, but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines, we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease. Highlights: ► A system for efficient and rapid genome editing with TALENs ► Generation of isogenic human cellular models of disease ► Identification of disease-associated phenotypes in multiple human cell types ► Minimal TALEN off-target effects, but significant clone-to-clone sequence variation Introduction: The study of human disease has been facilitated by the ability to identify the gene mutations responsible; at the same time, it has been hampered by the lack of an inexhaustible supply of easily accessible tissues from patients bearing those mutations. Another limitation is that many gene mutations that would be informative for disease biology if they could be studied in isolated cells are incompatible with human life (i.e., embryonic lethal). Classical gene-targeting technology via homologous recombination has proven to be an invaluable tool of experimental biology through its use in mouse embryonic stem cells for generating germline knockout and knockin mice; however, its use in mammalian systems has been limited primarily to studies in mice. In many cases, mice do not faithfully phenocopy human physiology and disease, e.g., cholesterol metabolism, coronary artery disease, and human hepatitis C virus (HCV) infection. The emergence of genome editing with engineered nucleases, as well as human pluripotent stem cell (hPSC) technology and differentiation protocols to obtain a variety of cell and tissue types in vitro, now make it possible to rapidly interrogate the effects of genetic modification in otherwise isogenic human model systems. Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that, due to their modular domain structure, have proven more straightforward to design and construct for performing genome editing than other types of nucleases (Bogdanove and Voytas, 2011). TALENs are typically designed as a pair that binds to genomic sequences flanking a target site and generates a double-strand break, which is repaired by the cell using either homology-directed repair (HDR) or the error-prone process of nonhomologous end-joining (NHEJ) (Christian et al., 2010; Li et al., 2011; Miller et al., 2011; Hockemeyer et al., 2011). NHEJ can be exploited to introduce small insertions or deletions (indels) resulting in frameshift mutations that effectively knock out a protein-coding gene. An exogenously introduced double-stranded DNA or single-stranded DNA oligonucleotide (ssODN) can serve as a repair template for HDR to incorporate an alteration into the genome (Soldner et al., 2011). In principle, TALEN pairs can be generated de novo with standard molecular biology techniques in a matter of days (Cermak et al., 2011; Sanjana et al., 2012). To demonstrate the utility, efficiency, and rapidity of TALEN technology in generating human cellular models with which to derive new biological insights, we created mutations in 15 genes and performed detailed phenotypic analysis of four genes for which novel roles in disease biology have emerged in recent years—APOB, SORT1, AKT2, and PLIN1. Results: Modular Assembly and Use of TALENs for Efficient and Rapid Genome Editing The DNA-binding domain of a TALEN comprises an array of 33- to 35-amino-acid monomers that are “coded” to recognize and bind specific DNA base pairs (bp) in a 1:1 fashion (Moscou and Bogdanove, 2009; Boch et al., 2009). We built upon previously described modular Golden Gate methodologies to allow assembly of multiple DNA fragments in an ordered fashion (Li et al., 2011; Cermak et al., 2011), such that a single ligation of preassembled tetramers and trimers generates TALENs that recognize any 15 bp recognition site in the genome (Figure 1; Figure S1 available online). This assembly method requires only 1–2 days for completion and is not prone to errors that complicate methods that rely on PCR amplification of monomers. Furthermore, we have developed a set of optimized vectors and methods for the delivery of TALENs into mammalian cells and, in particular, hPSCs. In brief, we transfect or electroporate TALEN pairs into cells and then subject them to fluorescence-activated cell sorting (FACS) 48 hr posttransfection based on fluorescent-marker expression. We replate the sorted cells at low density and allow them to recover and grow for 1 week, resulting in the formation of distinct single colonies. Colonies are expanded, genomic DNA purified, and mutations analyzed by PCR, agarose-gel screening, and Sanger sequencing (Figure 1 and Figure S1). The entire process from start to finish can be completed in less than one month., National Institutes of Health (U.S.) (R01-DK097768)

Recent advances in nuclear reprogramming technology allow the transformation of terminally differentiated, adult cells into induced pluripotent stem cells whose phenotype is indistinguishable from that of embryonic stem cells. This leap forward enables the creation of patient-specific pluripotent cell lines that carry disease genotypes. These cell lines could be used both as in vitro models for the study of disease and as potential sources of material for cell replacement therapy. Ultimately, a greater understanding of the process by which cellular identity is shaped and altered may allow the generation of particular cell types for the treatment of degenerative disease.

SummaryTissue culture of immortal cell strains from diseased patients is an invaluable resource for medical research but is largely limited to tumor cell lines or transformed derivatives of native tissues. Here we describe the generation of induced pluripotent stem (iPS) cells from patients with a variety of genetic diseases with either Mendelian or complex inheritance; these diseases include adenosine deaminase deficiency-related severe combined immunodeficiency (ADA-SCID), Shwachman-Bodian-Diamond syndrome (SBDS), Gaucher disease (GD) type III, Duchenne (DMD) and Becker muscular dystrophy (BMD), Parkinson disease (PD), Huntington disease (HD), juvenile-onset, type 1 diabetes mellitus (JDM), Down syndrome (DS)/trisomy 21, and the carrier state of Lesch-Nyhan syndrome. Such disease-specific stem cells offer an unprecedented opportunity to recapitulate both normal and pathologic human tissue formation in vitro, thereby enabling disease investigation and drug development.

Human pluripotent stem cells (hPSC) hold great promise as models for understanding disease and as a source of cells for transplantation therapies. However, the lack of simple, robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a platform for the culture of hPSCs that 1) allows for dissociation and replating of single cells, 2) significantly increases viability and replating efficiency, 3) improves freeze/thaw viability 4) improves cloning efficiency and 5) colony size variation. When combined with standard methodologies for genetic manipulation, we found that the enhanced culture platform allowed for lentiviral transduction rates of up to 95% and electroporation efficiencies of up to 25%, with a significant increase in the total number of antibiotic-selected colonies for screening for homologous recombination. We further demonstrated the utility of the enhanced culture platform by successfully targeting the ISL1 locus. We conclude that many of the difficulties associated with culturing and genetic manipulation of hPSCs can be addressed with optimized culture conditions, and we suggest that the use of the enhanced culture platform could greatly improve the ease of handling and general utility of hPSCs.