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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We describe a protocol for extracting high-quality nuclei from cryopreserved induced pluripotent stem cell-derived stromal/endothelial and blood cell types to support single-nucleus next-generation sequencing analyses. Producing high-quality, intact nuclei is imperative for multiomics experiments but can be a barrier to entry in the field for some laboratories.

Abstract

Induced pluripotent stem cell (iPSC)-based models are excellent platforms to understand blood development, and iPSC-derived blood cells have translational utility as clinical testing reagents and transfusable cell therapeutics. The advent and expansion of multiomics analysis, including but not limited to single nucleus RNA sequencing (snRNAseq) and Assay for Transposase-Accessible Chromatin sequencing (snATACseq), offers the potential to revolutionize our understanding of cell development. This includes developmental biology using in vitro hematopoietic models. However, it can be technically challenging to isolate intact nuclei from cultured or primary cells. Different cell types often require tailored nuclear preparations depending on cellular rigidity and content. These technical difficulties can limit data quality and act as a barrier to investigators interested in pursuing multiomics studies. Specimen cryopreservation is often necessary due to limitations with cell collection and/or processing, and frozen samples can present additional technical challenges for intact nuclear isolation. In this manuscript, we provide a detailed method to isolate high-quality nuclei from iPSC-derived cells at different stages of in vitro hematopoietic development for use in single-nucleus multiomics workflows. We have focused the method development on the isolation of nuclei from iPSC-derived adherent stromal/endothelial cells and non-adherent hematopoietic progenitor cells, as these represent very different cell types with regard to structural and cellular identity. The described troubleshooting steps limited nuclear clumping and debris, allowing the recovery of nuclei in sufficient quantity and quality for downstream analyses. Similar methods may be adapted to isolate nuclei from other cryopreserved cell types.

Introduction

Hematopoiesis is a relatively well-characterized developmental system, but an inability to recapitulate blood cell formation in vitro demonstrates an incomplete understanding of related factors. Induced pluripotent stem cell (iPSC)-based hematopoiesis models can help elucidate key developmental factors and related biology. The iPSC system also offers an excellent model to study blood disorders, and iPSC-based blood cells have been developed to produce translationally and therapeutically relevant reagents1,2,3,4. Single-cell studies ....

Protocol

1. Cryopreservation of cells

  1. Freeze >1 x 106 isolated iPSC-derived cells per mL in 1 mL of 90% FBS and 10% DMSO. Put the cryovials into a freezing container at -80°C to reduce the speed of freezing and optimize viable cell recovery (Table of Materials). Anticipate considerable cell loss, which can vary based on culture conditions and cell identity.
  2. Move from -80 °C to liquid nitrogen storage within 24 h.

Representative Results

We employed the aforementioned protocol to extract nuclei from cryopreserved iPSC-derived adherent stromal/endothelial cells and non-adherent (floating) hematopoietic progenitor cells. A detailed schematic representation of the nuclear isolation procedure can be found in Figure 1.

For morphological examination, isolated nuclei were stained with trypan blue and visualized under a microscope. Nuclear morphologic examination is critical immediately prior to processin.......

Discussion

Critical steps within the protocol
Isolating high quality nuclei is essential for the successful implementation of current single nucleus-based next generation sequencing modalities, which are rapidly evolving. In recognition of existing barriers for labs interested in these approaches, particularly for cryopreserved specimens, our intention was to craft a nuclear isolation technique tailored for previously frozen cells. Isolating nuclei from cryopreserved specimens is often necessary for clinical .......

Acknowledgements

The authors thank Jason Hatakeyama (10x Genomics) and Diana Slater (Children's Hospital of Philadelphia Center for Applied Genomics) for guidance and suggestions. This study was supported by the National Institutes of Health (HL156052 to CST).

....

Materials

NameCompanyCatalog NumberComments
Cell Culture Reagents
Dimethyl sulfoxide solution (DMSO)Sigma673439
Dulbecco's Phosphate-Buffered Saline (DPBS)Corning21031CV
Fetal Bovine Serum (FBS)GeminiBio100106
RPMI Medium 1640 (1X)Gibco11875093
Cells
Induced pluripotent stem cellsN/AN/AThe iPSCs used in this study were obtained through the CHOP Human Pluripotent Stem Cell Core Facility
Cell Staining Reagents 
Trypan Blue SolutionCorning25900CI
Dead Cell Removal Reagents
Calcium chloride (CaCl2)SigmaC4901
EasySep Dead Cell Removal (Annexin V) KitStemCell Technologies17899This kit is designed for the depletion of unwanted cell types labeled with biotin. The kit includes Dead Cell Removal (Annexin V) Cocktail, Biotin Selection Cocktail, and Dextran beads. This kit targets phosphatidylserine on the outer leaflet of the cell membrane of apoptotic cells using Annexin V. Unwanted cells are labeled with Annexin V, Biotinylated antibodies, and magnetic particles, and separated without columns using an EasySep magnet. Desired cells are simply poured off into a new tube.
Materials
10 µl MicropipetteWards science470231606
100 µl MicropipetteWards science470231598
1000 µl Extended Universal TipOxford Lab ProductsOAR-1000XL-SLF
1000 µl MicropipetteWards science470231602
2.0 ml Cryogenic vialsCorning431386
20 µl MicropipetteWards science470231608
200 µl MicropipetteWards science470231600
5ml Polystyrene Round-Bottom TubeFalcon352063
Corning DeckWork 0.1-10 µl Pipet Tip StationCorning4143
Corning DeckWork 1-200 µl Pipet Tip StationCorning4144
Counting chamberCytoSMART699910591
Flowmi Cell Strainer, 40 µm Bel-ArtH136800040
MagnetStemCell Technologies18000
NALGENE Cryo 1°C Freezing ContainerNalgene51000001
Nuclei Isolation ReagentsNuclei isolation reagents include Lysis Buffer,  Wash buffer, and Diluted Nuclei Buffer,and Lysis Dilution Buffer. The constitution of these buffers are in the Table 1, 2, and 3. Our nuclei isolation method improved isolation from the standard kit protocols (e.g., 10x Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide [CG000338]). This protocol can be used with several commercial kits, including the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle, 16 rxns (PN-1000283).
Dead Cell Removal kitSTEMCELL17899
DigitoninThermo Fisher ScientificBN2006
DL-Dithiothreitol solution (DTT)Sigma646563
Flowmi Cell Strainer, 40 µmBel-ArtH136800040
MACS bovine serum albumin (BSA) stock SolutionMiltenyi Biotec130091376
Magnesium chloride (MgCl2)Sigma7786303
Magnesium Chloride Solution, 1 MSigmaM1028
Nonidet P40 SubstituteSigma74385
Nuclease-Free WaterThermo Fisher ScientificAM9937
Nuclei Buffer (20X)10X Genomics2000153
Protector RNase inhibitorSigma3335399001
Sodium chloride (NaCl)SigmaS7653-250G
Sodium Chloride Solution, 5 MSigma59222C
Trizma Hydrochloride Soultion, pH 7.4SigmaT2194
Trizma hydrochloride (Tris-HCl) (pH7.4)SigmaT3252-500G
Tween 20Bio-Rad1662404
Other equipment
Automated cell counterCytoSMART699910591
MicroscopeZeissPrimostar 3

References

Explore More Articles

Nuclear IsolationCryopreservationIn Vitro Derived Blood CellsInduced Pluripotent Stem CellsIPSCSingle nucleus MultiomicsHematopoietic DevelopmentStromal CellsEndothelial CellsHematopoietic Progenitor Cells

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