This work was funded in part by NIH/NIDDK (R01DK096239) and New York State Stem Cell Science (NYSTEM C029156). Z.Z. was supported by the NYSTEM postdoctoral fellowship from the Center for Stem Cell Biology of the Sloan Kettering Institute.

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The authors have nothing to disclose.

Time Considerations for Generating Mutant Lines
Although recent approaches based on CRISPR/Cas systems for genome editing have led to successful targeting, a more efficacious and universal platform would be preferable for larger-scale analyses of gene function. The iCRISPR platform offers a rapid and efficient method to introduce mutations to any gene of interest5,9. First, the PCR-based gRNA synthesis method allows the production of hundreds of gRNAs in arrayed format in one day without the time-consuming cloning steps. Second, with doxycycline-inducible Cas9 expression in iCas9 hPSCs, the step involving gRNA transfection requires only a minimal amount of work, and thus, multiple gRNA targeting experiments can be conducted concurrently. Third, due to the high targeting efficiencies attainable with our system, the analysis of ~ 24 - 48 colonies per gRNA transfected should be sufficient to establish multiple monoallelic and biallelic mutant lines for a single gene, although efficiencies vary depending on the target locus. Since it is feasible for a trained individual to mechanically pick 384 colonies (4 x 96-well plates) in one sitting, which should take ~ 4 h under a dissecting microscope, a trained individual can be expected to generate mutant lines affecting 12 genes within 1 - 2 months. The streamlined generation of hPSC mutants in a short time allows for the systematic analysis of an array of transcription factors and/or signaling pathway components that interact with each other, regulating the developmental process9. Additionally, efficient multiplexed gene targeting also opens the door to investigating genetic interactions underlying complex human traits.
Generation of Precise Genetic Modifications
The derivation of patient-specific iPSCs from easily accessible somatic cell types and the differentiation into disease-relevant cell types provide a great opportunity for the functional validation of disease-associated mutations. However, due to the considerable variability in genetic background between individuals, direct comparisons between iPSCs from patients and from healthy donors may not allow one to distinguish disease phenotypes from background effects. Therefore, it is necessary to generate isogenic control iPSCs by correcting the disease mutation back to the wild-type sequence or to introduce the patient-specific mutations into a wild-type hPSC background, as proposed by others25,26. In addition to loss-of-function (null) mutations, one may now more precisely dissect disease mechanisms through the introduction of a patient-specific sequence alterations into the endogenous locus in hPSCs, including hypermorphic, hypomorphic, neomorphic, or dominant-negative patient mutations.
Precise genetic modification employs HDR for DSB repair in the presence of a repair template. Since it is much less efficient than NHEJ-mediated DNA repair, a donor plasmid containing the patient-specific mutation, a drug selection cassette, and homology arms has been previously used as the repair template2. After drug selection and verification of the patient-specific mutation, a second step is generally needed to remove the drug selection cassette. While the most widely used Cre-loxP and FLP-FRT systems leave behind residual sequence in the endogenous locus, the use of piggyBac transposon has allowed for the seamless removal of the drug selection cassette27. More recently, short ssDNA templates have also been shown to support efficient HDR with engineered DNA endonucleases28. Compared to a donor plasmid, ssDNA can be directly synthesized and thus circumvents the time-consuming cloning step. Here, it has been shown that, by co-transfecting gRNA and an ssDNA template to induce homology-directed repair, iCRISPR can be used to introduce specific nucleotide modifications with high efficiency. This is critical for not only dissecting the role of essential nucleotides within protein functional domains, but also for modeling human disease mutations and potentially correcting these disease-associated mutations for therapeutic intervention. Due to the large number of susceptibility loci that are each associated with multiple sequence variants, modeling complex and multigenic diseases such as diabetes has been a challenge for geneticists. As the iCRISPR platform is permissive for the rapid generation of an allelic series or for multiplexable gene targeting, it can facilitate the investigation of multiple disease-associated loci, either individually or in combination with isogenic backgrounds.
Targeting Efficiencies and Off-target Effects
We have found a good correlation between the T7E1 and RFLP assay results and the number of mutant lines identified by sequencing. This emphasizes the importance of performing these assays in parallel with the establishment of clonal lines. While the targeting efficiencies attained may vary depending on the genomic loci, in most single-gene-targeting experiments, 20 - 60% of the clones were found with both alleles mutated (including in-frame and frameshift mutations)9. In cases where multiplexed gene targeting was performed, triple biallelic mutant clones with 5-10% efficiencies were obtained5. ssDNA-mediated HDR of several genes were also performed to obtain precise genetic alterations, with the efficiencies of obtaining homozygous knock-in clones ranging from 1 - 10%5. Working with CRISPR/Cas in hPSCs, any mutations in potential off-target sites that do not share the same gRNA target sequence are yet to be detected5,9,12. Whole-genome sequencing performed in a recent study has also failed to identify substantial off-target mutations in clonal hPSC lines generated using CRISPR/Cas29. Nevertheless, to minimize any potential effect of confounding phenotypes introduced by mutations at off-target effect sites, it is suggested to generate independent mutant lines using at least two independent gRNAs targeting different sequences within the same gene. Similar phenotypes observed in multiple lines generated using different gRNAs could principally rule out the possibility that the phenotype comes from an off-target effect.
Feeder-dependent versus Independent Culture and Targeting
Traditional methods for hPSC culture involve their maintenance and expansion on feeder cells in media containing serum or serum replacement that includes animal products, such as bovine serum albumin. Feeder cells, serum, serum replacement, and albumin all contain complex, undefined components and show considerable batch variability. Adapting to the feeder-free and chemically defined condition significantly reduces the efforts for hPSC maintenance and, more importantly, increases the consistency of differentiation experiments. At present, there are very few studies that describe genome editing procedures on hPSCs cultured in fully defined culture conditions30. We have found higher CRISPR targeting efficiencies when gRNA transfection and clonal deposition were performed in feeder-free conditions compared to feeder-dependent culture conditions. We believe that this is due to increased cell survival after transfection and single-cell seeding for colony formation. Moreover, feeder cells have previously been shown to sequester transfection reagents, thereby lowering the transfection efficiency.
Combining Genome Editing with Directed Differentiation
Previous differentiation protocols have yielded only a small fraction of insulin-positive cells, the majority of which were polyhormonal and resembled fetal endocrine cells23. Recent progress has permitted the differentiation of hPSCs into more mature glucose-responsive beta-like cells16,17,19,20. We can routinely obtain at least 75% definitive endoderm cells, 40% PDX1+NKX6.1+ pancreatic progenitors, and around 20% NKX6.1+CPEP+ glucose-responsive beta-like cells using HUES8 hPSCs9. When combined with the iCRISPR genome editing system, this more robust differentiation protocol has facilitated the analysis of transcription factors that are crucial to the pancreatic progenitor and endocrine stages of pancreatic differentiation. This will make it possible, in future studies, to examine a large number of candidate disease genes for the functional validation and investigation into the mechanisms that underlie diabetes9.
Future Applications or Directions
Our iCRISPR system may facilitate the generation of more complex genomic modifications, such as the creation of reporter alleles through HDR-mediated gene targeting using long donor DNA templates encoding protein tags or fluorescent reporters12. We have shown that, due to the high CRISPR targeting efficiencies attained in the system, this process can be performed in hPSCs, without the need for further drug selection12.&#160; Furthermore, multiplexed gene targeting can be used to investigate genetic interactions underlying complex human disease, as shown in our recent study, which we believe is the first example of such work31. iCRISPR can also be used to understand gene regulatory control by creating deletions either in noncoding RNAs or in gene regulatory regions, such as promoters and enhancers. To efficiently generate regulatory mutants using iCRISPR, gRNAs can be designed to disrupt the binding site of a DNA-binding protein, including but not restricted to the basal transcriptional machinery or a tissue-specific transcription factor. ssDNA template-mediated HDR can also be used to mutate specific protein-binding sites. Finally, we envisage that further optimization would enable the use of the iCRISPR platform in hPSCs for higher throughput genetic analyses of pluripotency phenotypes or disease phenotypes when combined with an in vitro differentiation protocol. These iCRISPR-mediated studies may allow for the more rapid identification of candidate disease-associated genes and studies of their functional relevance.

Human pluripotent stem cells (hPSCs) have the ability to both self-renew and give rise to all derivatives of the three embryonic germ lineages. They provide a valuable resource for cell replacement therapy and disease modeling by serving as a unique platform to recapitulate cellular processes in a human developmental context. They are also a source of experimental cells for scalable, high-throughput analyses. However, progress has been limited because of two main challenges: the lack of efficient genetic modification tools and the difficulty in recapitulating the complex embryonic developmental steps in a culture dish.
Genetic modification is an indispensable tool to study gene function in normal development and disease. However, while classical gene targeting approaches via homologous recombination have proven to be a powerful tool to dissect gene function in mouse embryonic stem cells (mESCs)1, this approach has been extremely inefficient when applied to hPSCs2,3. The recent brisk accession of programmable, site-specific nucleases from nature to laboratory use, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems4, means that genome engineering has become a much easier task in a wide range of organisms and cell lines, including in hPSCs. These gene editing tools take advantage of the fact that chimeric nucleases such as the Cas9 endonuclease can permit a whole range of genetic modifications by inducing double-stranded breaks (DSBs) at precise locations, triggering the endogenous DNA repair machinery to activate either non-homologous end joining (NHEJ) or homology-directed repair (HDR). Both mechanisms may be exploited for genetic manipulation by inducing either random insertion and deletion mutations (indels; via NHEJ), to create frameshift mutations that nullify gene alleles, or precise nucleotide substitutions (via HDR), to recapitulate patient mutations for human disease modeling or to correct a disease-causing mutation for gene therapy.
CRISPR/Cas-mediated genome engineering requires two components: the constant RNA-guided Cas9 endonuclease necessary for DNA cleavage and a variable CRISPR RNA (crRNA) and trans-activating (tracrRNA) duplex that specifies DNA target recognition. The crRNA/tracrRNA duplex can be replaced with a single chimeric guide RNA (gRNA), which have been found to work more efficiently5,6,7. While the CRISPR/Cas9 system has been adapted to most experimental organisms and cell lines, the delivery and expression of Cas9 and gRNA varies significantly and needs to be further optimized to achieve efficient genome editing in many systems, including hPSCs8. An efficient genome-editing platform, iCRISPR, has been established in hPSCs5. In this system, a TALEN-mediated approach has been used to target both alleles of the &#34;transgene safe harbor locus&#34; AAVS1 in trans, one allele with a reverse tetracycline-controlled transactivator (M2rtTA) and the other with a tetracycline response element (TRE) driving the expression of Cas9 (iCas9) in the hPSCs. In established clonal lines (iCas9 hPSCs), Cas9 is highly expressed with doxycycline treatment. Meanwhile, due to its small size (100 nt), single or multiple gRNAs can be easily delivered into iCas9 hPSCs with high efficiency and can direct Cas9 for site-specific cleavage, enabling efficient NHEJ-mediated gene disruption, as well as HDR-mediated precise nucleotide modifications in the presence of short single-stranded DNA (ssDNA) donor templates. The iCRISPR system can be used to successfully generate a panel of disease-mimicking hPSC lines with biallelic (homozygous or compound heterozygous) or heterozygous loss-of-function mutations in important developmental genes5,9. While a number of groups have reported efficient gene editing using CRISPR/Cas in hPSCs, the success remains limited to a small number of technologically adept laboratories. The iCRISPR platform offers an efficient yet simple solution for routine gene editing by researchers of different skill levels, and it has already been used in a number of published studies by our group and others9,10,11,12. This approach has also been further extended to inducible silencing based on the expression of dCas9-KRAB13.
Along with the progress in genome-editing technology, significant improvements have also been achieved in hPSC maintenance and directed differentiation. Culture conditions for hPSCs have evolved from irradiated mouse embryonic fibroblast (iMEF) feeder-dependent to feeder-free conditions on defined extracellular matrix components, and from complex media formulations to chemically defined medium conditions14. Such improvements have reduced the variability in hPSCs due to batch-to-batch differences in iMEF preparation and knockout serum replacement components, and thus provide a more reproducible environment for hPSC differentiation. Meanwhile, improved knowledge of signaling pathways governing human embryonic development, as well as discoveries from high-throughput drug screenings, have led to improved differentiation protocols15,16,17,18. These protocols more closely mimic in vivo developmental steps and generate cell types that closely recapitulate their in vivo counterparts. For hPSC differentiation into the pancreatic lineage, initial protocols mimicked early pancreatic development relatively well but eventually generated polyhormonal &#946; cells that were of immature fetal phenotypes and responded poorly to glucose stimulation. Recent advancements16,17,19,20 have allowed for the generation of glucose-responsive pancreatic &#946;-like cells, which will enable us to investigate later events, such as the formation and the further maturation of monohormonal &#946; cells.
Here, we detail the application of genome-modified lines for the study of pancreatic development by combining the iCRISPR system with the hPSC-based in vitro differentiation platform towards glucose-responsive pancreatic &#946;-like cells. This coupling of powerful genome editing tools with an improved hPSC differentiation protocol not only offers the speed and scale necessary to meet the growing demand for validating disease causality, but also enables sophisticated genetic manipulations for further mechanistic investigations into transcriptional control underlying normal development and disease9.

<table><tbody><tr><td>Chemically defined medium (E8)</td><td>Thermo Fisher Scientific</td><td>A1517001</td><td>Essential 8 basal medium and Essential 8 supplement included</td></tr><tr><td>Truncated recombinant human form of vitronectin</td><td>Thermo Fisher Scientific</td><td>A14700</td><td></td></tr><tr><td>ROCK inhibitor Y-27632</td><td>Selleck Chemicals</td><td>S1049</td><td></td></tr><tr><td>Dissociation reagent (TrypLE Select enzyme)</td><td>Thermo Fisher Scientific</td><td>12563029</td><td>1x, animal origin free, recombinant enzyme</td></tr><tr><td>G418 Sulfate</td><td>Thermo Fisher Scientific</td><td>10131035</td><td>Geneticin Selective Antibiotic</td></tr><tr><td>Puromycin dihydrochloride</td><td>Sigma-Aldrich</td><td>P8833</td><td>Puromycin Selective Antibiotic</td></tr><tr><td>DNA Polymerase (Herculase II Fusion)</td><td>Agilent Technologies</td><td>600679</td><td>PCR kit</td></tr><tr><td>MEGAshortscript T7 Transcription kit</td><td>Thermo Fisher Scientific</td><td>AM1354</td><td></td></tr><tr><td>MEGAclear Transcription Clean-Up Kit</td><td>Thermo Fisher Scientific</td><td>AM1908</td><td></td></tr><tr><td>Doxycycline hyclate</td><td>Sigma-Aldrich</td><td>D9891</td><td></td></tr><tr><td>Opti-MEM medium</td><td>Thermo Fisher Scientific</td><td>31985062</td><td>Reduced Serum Medium</td></tr><tr><td>Lipofectamine RNAiMAX Transfection Reagent</td><td>Thermo Fisher Scientific</td><td>13778150</td><td></td></tr><tr><td>DNeasy Blood &amp;amp; Tissue Kit</td><td>QIAGEN</td><td>69504</td><td>Genomic DNA extraction kit</td></tr><tr><td>Proteinase K</td><td>Roche</td><td>3115879001</td><td></td></tr><tr><td>MCDB 131 medium</td><td>Thermo Fisher Scientific</td><td>10372-019</td><td></td></tr><tr><td>BLAR medium</td><td>Thermo Fisher Scientific</td><td></td><td>Custom-made with a published formulation (Rezania &lt;em&gt;et al.&lt;/em&gt;, 2014)</td></tr><tr><td>L-glutamine supplement (GlutaMAX)</td><td>Thermo Fisher Scientific</td><td>35050061</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Thermo Fisher Scientific</td><td>144-55-8</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8769</td><td></td></tr><tr><td>BSA</td><td>LAMPIRE Biological Laboratories</td><td>7500855</td><td>Fatty acid free</td></tr><tr><td>GDF8</td><td>PeproTech</td><td>120-00</td><td></td></tr><tr><td>CHIR-99021</td><td>Stemgent</td><td>04-0004</td><td>GSK-3 inhibitor</td></tr><tr><td>L-Ascorbic acid</td><td>Sigma-Aldrich</td><td>A4544</td><td>Vitamin C</td></tr><tr><td>FGF7</td><td>R&amp;amp;D Systems</td><td>251-KG</td><td></td></tr><tr><td>SANT1</td><td>Tocris Bioscience</td><td>1974</td><td>Hedgehog inhibitor</td></tr><tr><td>RA</td><td>Sigma-Aldrich</td><td>R2625</td><td>Retinoic acid</td></tr><tr><td>LDN</td><td>Stemgent</td><td>04-0019</td><td>BMP inhibitor</td></tr><tr><td>IWP-2</td><td>Tocris Bioscience</td><td>3533</td><td>Wnt antagonist&amp;nbsp;</td></tr><tr><td>ITS-X</td><td>Thermo Fisher Scientific</td><td>51500-056</td><td></td></tr><tr><td>TPB</td><td>EMD Millipore</td><td>565740-1MG</td><td>PKC activator</td></tr><tr><td>3,3&amp;rsquo;,5-Triiodo-L-thyronine (T3)</td><td>Sigma-Aldrich</td><td>T6397</td><td></td></tr><tr><td>ALK5i II</td><td>Enzo Life Sciences</td><td>ALX-270-445</td><td>ALK5 inhibitor II</td></tr><tr><td>ZnSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>Z0251</td><td></td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H3149</td><td></td></tr><tr><td>GSiXX</td><td>EMD Millipore</td><td>565789</td><td>Gamma secretase inhibitor XX, NOTCH signaling inhibitor</td></tr><tr><td>N-Cys (N-acetyl cysteine)</td><td>Sigma-Aldrich</td><td>A9165</td><td></td></tr><tr><td>Trolox</td><td>EMD Millipore</td><td>648471</td><td>Vitamin E analogue</td></tr><tr><td>R428</td><td>Selleck Chemicals</td><td>S2841</td><td>AXL receptor tyrosine kinase inhibitor</td></tr><tr><td>24 mm Transwell with insert</td><td>Corning Life Sciences</td><td>3414</td><td></td></tr><tr><td>Gene Pulser Xcell Electroporation System</td><td>Bio-Rad</td><td>1652660</td><td></td></tr><tr><td>0.4 cm Electroporation Cuvettes</td><td>Bio-Rad</td><td>1652081</td><td></td></tr><tr><td>AAVS1-TALEN-L</td><td>Addgene</td><td>59025</td><td></td></tr><tr><td>AAVS1-TALEN-R</td><td>Addgene</td><td>59026</td><td></td></tr><tr><td>AAVS1-Neo-M2rtTA</td><td>Addgene</td><td>60843</td><td></td></tr><tr><td>AAVS1-Puro-iCas9</td><td>Addgene</td><td>58409</td><td></td></tr><tr><td>T7 Endonuclease I</td><td>NEB</td><td>M0302L</td><td></td></tr><tr><td>Buffer 2</td><td>NEB</td><td>B7002S</td><td>NEBuffer 2</td></tr><tr><td>Long oligonucleotide</td><td>Eton Bioscience or IDT</td><td></td><td></td></tr><tr><td>SOX17 antibody</td><td>R&amp;amp;D Systems</td><td>AF1924</td><td>1:500</td></tr><tr><td>FOXA2 antibody</td><td>Millipore</td><td>07-633</td><td>1:100</td></tr><tr><td>CXCR4-APC antibody</td><td>R&amp;amp;D Systems</td><td>FAB170A</td><td>1:25</td></tr><tr><td>PDX1 antibody</td><td>R&amp;amp;D Systems</td><td>AF2419</td><td>1:500</td></tr><tr><td>NKX6.1 antibody</td><td>DSHB</td><td>F55A12</td><td>1:500</td></tr><tr><td>NKX2.2 antibody</td><td>DSHB</td><td>74.5A5</td><td>1:100</td></tr><tr><td>NEUROD1 antibody</td><td>Santa Cruz Biotechnology</td><td>sc-1084</td><td>1:100</td></tr><tr><td>Insulin antibody</td><td>Dako</td><td>A0564</td><td>1:2,000</td></tr><tr><td>C-peptide antibody</td><td>DSHB</td><td>GN-ID4-c</td><td>1:2,000</td></tr><tr><td>Glucagon antibody</td><td>Sigma-Aldrich</td><td>G2654</td><td>1:1,000</td></tr></tbody></table>

genome editing and directed differentiation of hpscs for interrogating lineage determinants in human pancreatic development

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic &#946;-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Watch this Scientific Journal Video about Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development at JoVE.com

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive &#946;-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding ...

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Research

JoVE Journal

Genetics

13.1K Views.  Sloan Kettering Institute.  Protocollen bij HPSC mutant lijnen met behulp van de iCRISPR platform te genereren en om hPSCs in glucose-responsief β-achtige cellen te onderscheiden zijn beschreven. De combinatie van genoom editing-technologie met HPSC gerichte differentiatie biedt een krachtig platform voor de systematische analyse van de rol van de lijn determinanten in de menselijke ontwikkeling en de progressie van de ziekte. 

Video: Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

Developmental Biology

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Bioengineering

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling,&#160;however,&#160;with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell&#39;s physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to ...

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 &#181;m diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish.

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and ...

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages 2, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3 in combination with several growth factors 4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine 8. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11.
The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.

The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are ...

Biology

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

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