Producing High Quality CRISPR-Cas9 Edited hiPSCs for Cardiac Disease Modeling

Maksymilian Prondzynski, PhD, Instructor, The Department of Cardiology, Boston Children’s Hospital and Harvard Medical School

What You Will Learn:
  • Quality criteria for human induced pluripotent stem cells (iPSCs) used for disease modeling experiments
  • Quality assessment and validation of human iPSCs after CRISPR editing
  • Reproducible cardiac differentiation using a stirred suspension bioreactor system
  • Methods of in vitro phenotyping using human iPSC-derived cardiomyocytes
Record Date: June 28, 2023

The Nobel prize winning discovery of human induced pluripotent stem cells (hiPSCs) facilitated a new era of disease modeling in vitro. This event inspired clinicians and scientists alike to use those cells for regenerative medicine, or for modeling of genetic diseases, to gain further insights into disease mechanisms on a cellular level. However, experimental studies using hiPSCs can experience high inter- and intra-experimental variability further fueling the reproducibility crisis in life sciences.

In the past 10 years much progress has been made in defining quality criteria (QC) for hiPSCs to achieve genome stability and high pluripotency over prolonged culture duration. These QC parameters include single cell clonal selection, karyotyping, pluripotency assessment and the establishment of master cell banks, especially in the context of CRISPR-Cas9 genome edited hiPSCs. Additionally, Numerous cardiac differentiation protocols have been established using monolayer (ML) adherent or 3D suspension cultures. However, generating large numbers of high quality human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with high consistency between batches, different cell lines and cryo-storage has remained challenging leading to poor reproducibility of experimental results.

The featured speakers used a stirred suspension bioreactor system to create an optimal environment for cell growth and differentiation. This enabled them to create a unified differentiation protocol that can be applied to a variety of hiPSCs (patient and control lines) with high cardiomyocyte content and reduced batch-to-batch variability. Using a wide array of cardiac disease modeling assays, they then confirmed high reproducibility of cryo-preserved hiPSC-derived cardiomyocytes.

Join this webinar to learn about optimizing the growth and differentiation of human induced pluripotent stem cells (hiPSCs) into cardiomyocytes and enhancing cardiac disease modeling.

Enriching High Titer IgG-Producing Clones Using Cold-Capture and The Namocell Pala Single Cell Dispenser

Francisco Ramirez, Manager, Application Science, Bio-Techne

What You Will Learn:
  • The conventional method of screening CHO cells for clones expressing high titer of monoclonal antibody and its challenges
  • The cold-capture technique that allows labeling of cells expressing secreted recombinant proteins
  • The potential of cold-capture-facilitated single cell cloning using a fully automated single cell dispenser to improve the efficiency in identifying high yield clones
Record Date: May 2, 2023

Cloning of Chinese hamster ovary (CHO) cells expressing recombinant antibodies is a crucial step in the development of monoclonal antibody (mAb) therapeutics. It is important to identify clones that produce high yield. However, the heterogeneity of CHO cells can give rise to varied IgG production among individual cells. Thus, the process of finding high titer clones typically involves screening of large numbers of clones, making it laborious and time-consuming.

Cold-capture is a technique that allows labelling of antibody producing cells with a fluorescent reagent that binds to the antibody molecules when they are transiently present on the cell membrane before release from secretory vesicles. This labelling could enable cell sorting based on the level of fluorescence signal to select higher productivity cells. In this study, the featured speaker sought to test the ability to enrich high titer IgG CHO cell clones by cold-capture, followed by isolation of single cells with the brightest fluorescent signal using the Namocell Pala Single Cell Dispenser — a fully automated dispenser that enables a fast, easy fluorescence and light scatter-based single cell isolation. The speaker will show that their method allowed the identification of clones with an average 3x higher IgG production from the cold-capture labelled cells compared to non-cold-captured cells.

These results suggest that the combination of cold-capture and single cell isolation could be an efficient and economical solution for improving cell line development in recombinant antibody production.

Watch this webinar to learn more about the potential of cold-capture-facilitated single cell isolation and cloning to improve the efficiency in identifying high yield clones.

Noninvasive Prenatal Testing: The Luna Prenatal Test for Isolation of Circulating Fetal Cells Early in Pregnancy

Mason Ouren, R&D Scientist, Luna Genetics

What You Will Learn:
  • Historic challenges to the establishment of a noninvasive cell-based prenatal test
  • How the Luna Prenatal Test isolates and analyzes single circulating fetal cells early in pregnancy
  • How the Luna Prenatal Test makes a positive difference in patient care and reproductive health
Record Date: March 17, 2023
Watch this webinar to learn about a breakthrough in the reliable isolation and successful analysis of circulating fetal cells for noninvasive prenatal testing early in pregnancy. The webinar will include the path taken to establish the Luna Prenatal Test methodology, data from key validation cases and the future trajectory of the test.

Chemical Cytoprotection for Stress-Free Use of Human Stem Cells in Basic and Translational Research

Dr. Ilyas Singec, Director, Stem Cell Translation Laboratory (SCTL), NIH Regenerative Medicine Program
What You Will Learn:
  • Quick and easy methods for isolating single cells for downstream applications
  • How to enhance cell survival of genetically stable human pluripotent stem cells (hPSCs)
  • How to ensure safe and robust stem cell growth with a novel, four-part small molecule cocktail
Record Date: November 9, 2021
Clinical translation of human pluripotent stem cells (hPSCs) requires advanced strategies that ensure safe and robust long-term growth and functional differentiation. We recently reported the development of a new small molecule cocktail that can be used as a universal end-to-end solution for stress-free culture of human cells. This four-part small molecule cocktail, which we named CEPT (Chroman 1, Emricasan, trans-ISRIB, Polyamines), enhanced cell survival of genetically stable hPSCs by simultaneously blocking several stress mechanisms that otherwise compromise cell structure and function. CEPT improved routine cell passaging, cryopreservation/thawing of pluripotent and differentiated cells, embryoid body and organoid formation, single-cell cloning, genome editing, and new iPSC line generation. Thus, CEPT represents a unique poly-pharmacological strategy for comprehensive cytoprotection, providing a new rationale for efficient and safe utilization of hPSCs. Conferring cell fitness by multi-target drug combinations may become a common approach in cryobiology, drug development, and regenerative medicine.

Fast, Gentle and Easy Single Cell Isolation

Mason Smith, Ph.D., Sr. Applications Scientist, Namocell
What You Will Learn:
  • Latest advancements in single cell sorting and dispensing technology
  • Discuss common pitfalls in several single cell workflows and practical tips to optimize results
  • Learn about novel single cell applications using innovative single cell sorting and dispensing technology
Record Date: October 7, 2021
Single cell sorting and isolation has traditionally been a bottleneck in the workflows for single cell cloning, single cell genomics, rare cell isolation, synthetic biology and other applications. Existing techniques such as fluorescence-activated cell sorting (FACS) often leads to compromised cell viability and poor outgrowth, contamination, clogging, in addition to the operational complexity and high maintenance requirement, while manual limiting dilution is inefficient, unreliable and highly labor-intensive. As such, there has been an urgent need for fast and easy single cell dispensing technology that is at the same time gentle to the cells and can preserve their viability and integrity. There is also a need for quick sorting of tens of thousands of cells for single cell genomics, such as sample prep for 10x Genomics workflow.

In this presentation, the speaker will discuss an innovative technology to address these needs. It combines the benefits of three key technologies: microfluidics, flow cytometry and liquid dispensing. This will simplify and empower a number of single cell applications, including cell line development, single cell genomics and proteomics, cell line engineering and CRISPR, monoclonal antibody development, synthetic biology, and rare cell isolation such as circulating tumor cells (CTCs) and circulating fetal cells.

Utilizing RNA and DNA Plate-Seq Workflows for Low-Input or Rare Single-Cell Genomics

Ioanna Andreou, Ph.D., Associate Director NGS & Applications Development, Qiagen
Felix Alonso-Valenteen, Ph.D., Field Application Scientist, Namocell
Applications: Single Cell Genomics
What You Will Learn:
  • Considerations, challenges and new technologies for low-input or rare single-cell genomics
  • Latest tips for low-input NGS
Record Date: April 26, 2022
Single-cell studies in recent years have revealed that organisms are composed of thousands of unique and unrepeatable cell types. Conducting single-cell genomic analyses using next generation sequencing (NGS) methods, similar to low-input sequencing, has traditionally been challenging due to the limited amount of genomic DNA present in single cells. This limitation becomes even more pronounced when the studied population is small, such as in the case of rare cells. High-fidelity (HiFi) and high-quality genomic DNA amplification is essential for single-cell sequencing, which is strongly dependent on the quality of the isolated cells. Therefore, the method used to isolate single cells becomes an important consideration for ensuring cell viability and nucleic acid integrity. Namocell’s patented gentle sorting and cell enrichment technology overcomes the aforementioned challenges to provide a higher quantity and quality of cell samples for sequencing. As a result, a complete and accurate picture of the studied samples can be achieved.