Evaluating Capture Sequence Performance for Single-Cell CRISPR Activation Experiments.

The combination of single-cell RNA sequencing with CRISPR inhibition/activation provides a high-throughput approach to simultaneously study the effects of hundreds if not thousands of gene perturbations in a single experiment. One recent development in CRISPR-based single-cell techniques introduces a feature barcoding technology that allows for the simultaneous capture of mRNA and guide RNA (gRNA) from the same cell. This is achieved by introducing a capture sequence, whose complement can be incorporated into each gRNA and that can be used to amplify these features prior to sequencing. However, because the technology is in its infancy, there is little information available on how such experimental parameters can be optimized. To overcome this, we varied the capture sequence, capture sequence position, and gRNA backbone to identify an optimal gRNA scaffold for CRISPR activation gene perturbation studies. We provide a report on our screening approach along with our observations and recommendations for future use.

EpiMogrify Models H3K4me3 Data to Identify Signaling Molecules that Improve Cell Fate Control and Maintenance.

The need to derive and culture diverse cell or tissue types in vitro has prompted investigations on how changes in culture conditions affect cell states. However, the identification of the optimal conditions (e.g., signaling molecules and growth factors) required to maintain cell types or convert between cell types remains a time-consuming task. Here, we developed EpiMogrify, an approach that leverages data from ∼100 human cell/tissue types available from ENCODE and Roadmap Epigenomics consortia to predict signaling molecules and factors that can either maintain cell identity or enhance directed differentiation (or cell conversion). EpiMogrify integrates protein-protein interaction network information with a model of the cell's epigenetic landscape based on H3K4me3 histone modifications. Using EpiMogrify-predicted factors for maintenance conditions, we were able to better potentiate the maintenance of astrocytes and cardiomyocytes in vitro. We report a significant increase in the efficiency of astrocyte and cardiomyocyte differentiation using EpiMogrify-predicted factors for conversion conditions.

Immobilization of vitronectin-binding heparan sulfates onto surfaces to support human pluripotent stem cells.

Functionalizing medical devices with polypeptides to enhance their performance has become important for improved clinical success. The extracellular matrix (ECM) adhesion protein vitronectin (VN) is an effective coating, although the chemistry used to attach VN often reduces its bioactivity. In vivo, VN binds the ECM in a sequence-dependent manner with heparan sulfate (HS) glycosaminoglycans. We reasoned therefore that sequence-based affinity chromatography could be used to isolate a VN-binding HS fraction (HS9) for use as a coating material to capture VN onto implant surfaces. Binding avidity and specificity of HS9 were confirmed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR)-based assays. Plasma polymerization of allylamine (AA) to tissue culture-treated polystyrene (TCPS) was then used to capture and present HS9 as determined by radiolabeling and ELISA. HS9-coated TCPS avidly bound VN, and this layered surface supported the robust attachment, expansion, and maintenance of human pluripotent stem cells. Compositional analysis demonstrated that 6-O- and N-sulfation, as well as lengths greater than three disaccharide units (dp6) are critical for VN binding to HS-coated surfaces. Importantly, HS9 coating reduced the threshold concentration of VN required to create an optimally bioactive surface for pluripotent stem cells. We conclude that affinity-purified heparan sugars are able to coat materials to efficiently bind adhesive factors for biomedical applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1887-1896, 2018.

Taking a Step Forward in Laparoscopic Donor Nephrectomy: Transvaginal Retrieval of Donor's Kidney.

Laparoscopic donor nephrectomy has been broadly recognized as the gold standard for kidney procurement used in kidney transplantation where it is not uncommon for donors to experience discomfort and aesthetic dissatisfaction over larger incision site. Natural orifice transluminal endoscopic surgery is a surgical approach that allows scarless intraabdominal operations through natural orifices, such as the vagina. In this case report, we describe the first case of transvaginal retrieval of donor's kidney at the National University Hospital, Singapore. A 51-year-old Malay lady with no significant medical history volunteered to a living-related kidney donor. Perioperative antibiotics were administered. A 12 mm Excel port was placed over the left iliac fossa with camera insertion. Two additional ports were inserted over the left rectus sheath edge and left costal margin under direct vision. An additional 5 mm port at the left loin was placed for lateral retraction. A vaginal probe was then inserted to facilitate posterior colpotomy and transection of the left uterosacral ligament. Pneumoperitoneum was subsequently maintained with a LiNA McCartney(®) Tube. A 15 mm Endocatch(®) bag was inserted for retrieval of the kidney. The left kidney was placed in the Endocatch bag after transection of the hilar vessels where the kidney was retrieved vaginally with ease. Colpotomy was closed vaginally using Vicryl-0 continuous suture. Total blood loss was noted as 50 mL with warm ischemia time being 7 minutes and the entire retrieval taking totally 20 minutes. Postoperative recovery was uneventful and the donor was discharged stable 3 days postoperation. The transplanted kidney retained normal graft function. Colpotomy retrieval for donor nephrectomy presents an innovative method for specimen retrieval with minimal disruption of donor anatomy. Doing away with laparotomy for kidney retrieval has indeed shown a reduction in recovery time, reduced postoperative pain, and better cosmetic outcome.

Microcarrier-Expanded Neural Progenitor Cells Can Survive, Differentiate, and Innervate Host Neurons Better When Transplanted as Aggregates.

Neuronal progenitor cells (NPCs) derived from human embryonic stem cells (hESCs) are an excellent cell source for transplantation therapy due to their availability and ethical acceptability. However, the traditional method of expansion and differentiation of hESCs into NPCs in monolayer cultures requires a long time, and the cell yield is low. A microcarrier (MC) platform can improve the expansion of hESCs and increase the yield of NPCs. In this study, for the first time, we transplanted microcarrier-expanded hESC-derived NPCs into the striatum of adult NOD-SCID IL2Rgc null mice, either as single cells or as cell aggregates. The recipient mice were perfused, and the in vivo survival, differentiation, and targeted innervation of the transplanted cells were assessed by immunostaining. We found that both the transplanted single NPCs and aggregate NPCs were able to survive 1 month posttransplantation, as revealed by human-specific neural cell adhesion molecule (NCAM) and human nuclear antigen staining. Compared to the single cells, the transplanted cell aggregates showed better survival over a 3-month period. In addition, both the transplanted single NPCs and the aggregate NPCs were able to differentiate into DCX-positive immature neurons and Tuj1-positive neurons in vivo by 1 month posttransplantation. However, only the transplantation of aggregate NPCs was shown to result in mature neurons at 3 months posttransplantation. Furthermore, we found that the cell aggregates were able to send long axons to innervate their targets. Our study provides preclinical evidence that the use of MCs to expand and differentiate hESC-derived NPCs and transplantation of these cells as aggregates produce longer survival in vivo.

Genome-Wide Transcriptome and Binding Sites Analyses Identify Early FOX Expressions for Enhancing Cardiomyogenesis Efficiency of hESC Cultures.

The differentiation efficiency of human embryonic stem cells (hESCs) into heart muscle cells (cardiomyocytes) is highly sensitive to culture conditions. To elucidate the regulatory mechanisms involved, we investigated hESCs grown on three distinct culture platforms: feeder-free Matrigel, mouse embryonic fibroblast feeders, and Matrigel replated on feeders. At the outset, we profiled and quantified their differentiation efficiency, transcriptome, transcription factor binding sites and DNA-methylation. Subsequent genome-wide analyses allowed us to reconstruct the relevant interactome, thereby forming the regulatory basis for implicating the contrasting differentiation efficiency of the culture conditions. We hypothesized that the parental expressions of FOXC1, FOXD1 and FOXQ1 transcription factors (TFs) are correlative with eventual cardiomyogenic outcome. Through WNT induction of the FOX TFs, we observed the co-activation of WNT3 and EOMES which are potent inducers of mesoderm differentiation. The result strengthened our hypothesis on the regulatory role of the FOX TFs in enhancing mesoderm differentiation capacity of hESCs. Importantly, the final proportions of cells expressing cardiac markers were directly correlated to the strength of FOX inductions within 72 hours after initiation of differentiation across different cell lines and protocols. Thus, we affirmed the relationship between early FOX TF expressions and cardiomyogenesis efficiency.

Inhibition of ROCK-myosin II signaling pathway enables culturing of human pluripotent stem cells on microcarriers without extracellular matrix coating.

Large quantities of human pluripotent stem cells (hPSCs) needed for therapeutic applications can be grown in scalable suspended microcarrier cultures. These microcarriers are coated with animal or human extracellular matrix (ECM) proteins to promote cell growth and maintain pluripotency. However, the coating is costly for large-scale cultures and it presents safety risks. This study demonstrates that hPSCs can be propagated on noncoated positively charged cellulose microcarriers in a serum-free medium containing the ROCK inhibitor, (Y27632) or myosin inhibitor, Blebbistatin. In the presence of these two inhibitors, myosin phosphatase 1 and myosin light chain 2 were dephosphorylated suggesting that reduced myosin contractility is responsible for hPSC survival and growth on ECM coating-free microcarriers. Cells propagated on the noncoated microcarriers for 12 passages maintained their pluripotency and karyotype stability. Scalability was demonstrated by achieving a cell concentration of 2.3×106 cells/mL with 11.5-fold expansion (HES-3) in a 100-mL spinner flask. The differentiation capability of these cells toward three primary lineages is demonstrated via in vitro embryoid bodies and in vivo teratoma formations. Moreover, the directed differentiation to polysialylated neuronal cell adhesion molecule-positive (PSA-NCAM+) neural progenitors produced high cell concentrations (9.1±1.2×106 cells/mL) with a cell yield of 412±77 neural progenitor cells per seeded HES-3 and a PSA-NCAM expression level of 91±1.1%. This defined serum- and coating-free scalable microcarrier culturing system is a safer and less expensive method for generating large amounts of hPSCs for cell therapies.

Microcarrier suspension cultures for high-density expansion and differentiation of human pluripotent stem cells to neural progenitor cells.

Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) can be differentiated to neural cells that model neurodegenerative diseases and be used in the screening of potential drugs to ameliorate the disease phenotype. Traditionally, NPCs are produced in 2D cultures, in low yields, using a laborious process that includes generation of embryonic bodies, plating, and colony selections. To simplify the process and generate large numbers of hiPSC-derived NPCs, we introduce a microcarrier (MC) system for the expansion of a hiPSC line and its subsequent differentiation to NPC, using iPS (IMR90) as a model cell line. In the expansion stage, a process of cell propagation in serum-free MC culture was developed first in static culture, which is then scaled up in stirred spinner flasks. A 7.7-fold expansion of iPS (IMR90) and cell yield of 1.3×106 cells/mL in 7 days of static MC culture were achieved. These cells maintained expression of OCT 3/4 and TRA-1-60 and possessed a normal karyotype over 10 passages. A higher cell yield of 6.1×106 cells/mL and 20-fold hiPSC expansion were attained using stirred spinner flasks (seeded from MC static cultures) and changing the medium-exchange regimen from once to twice a day. In the differentiation stage, NPCs were generated with 78%-85% efficiency from hiPSCs using a simple serum-free differentiation protocol. Finally, the integrated process of cell expansion and differentiation of hiPSCs into NPCs using an MC in spinner flasks yielded 333 NPCs per seeded hiPSC as compared to 53 in the classical 2D tissue culture protocol. Similar results were obtained with the HES-3 human embryonic stem cell line. These NPCs were further differentiated into ßIII-tubulin⁺ neurons, GFAP⁺ astrocytes, and O4⁺ oligodendrocytes, showing that cells maintained their multilineage differentiation potential.