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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.
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.
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 ....
1. Cryopreservation of cells
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.......
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 .......
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).
....Name | Company | Catalog Number | Comments |
Cell Culture Reagents | |||
Dimethyl sulfoxide solution (DMSO) | Sigma | 673439 | |
Dulbecco's Phosphate-Buffered Saline (DPBS) | Corning | 21031CV | |
Fetal Bovine Serum (FBS) | GeminiBio | 100106 | |
RPMI Medium 1640 (1X) | Gibco | 11875093 | |
Cells | |||
Induced pluripotent stem cells | N/A | N/A | The iPSCs used in this study were obtained through the CHOP Human Pluripotent Stem Cell Core Facility |
Cell Staining Reagents | |||
Trypan Blue Solution | Corning | 25900CI | |
Dead Cell Removal Reagents | |||
Calcium chloride (CaCl2) | Sigma | C4901 | |
EasySep Dead Cell Removal (Annexin V) Kit | StemCell Technologies | 17899 | This 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 Micropipette | Wards science | 470231606 | |
100 µl Micropipette | Wards science | 470231598 | |
1000 µl Extended Universal Tip | Oxford Lab Products | OAR-1000XL-SLF | |
1000 µl Micropipette | Wards science | 470231602 | |
2.0 ml Cryogenic vials | Corning | 431386 | |
20 µl Micropipette | Wards science | 470231608 | |
200 µl Micropipette | Wards science | 470231600 | |
5ml Polystyrene Round-Bottom Tube | Falcon | 352063 | |
Corning DeckWork 0.1-10 µl Pipet Tip Station | Corning | 4143 | |
Corning DeckWork 1-200 µl Pipet Tip Station | Corning | 4144 | |
Counting chamber | CytoSMART | 699910591 | |
Flowmi Cell Strainer, 40 µm | Bel-Art | H136800040 | |
Magnet | StemCell Technologies | 18000 | |
NALGENE Cryo 1°C Freezing Container | Nalgene | 51000001 | |
Nuclei Isolation Reagents | Nuclei 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 kit | STEMCELL | 17899 | |
Digitonin | Thermo Fisher Scientific | BN2006 | |
DL-Dithiothreitol solution (DTT) | Sigma | 646563 | |
Flowmi Cell Strainer, 40 µm | Bel-Art | H136800040 | |
MACS bovine serum albumin (BSA) stock Solution | Miltenyi Biotec | 130091376 | |
Magnesium chloride (MgCl2) | Sigma | 7786303 | |
Magnesium Chloride Solution, 1 M | Sigma | M1028 | |
Nonidet P40 Substitute | Sigma | 74385 | |
Nuclease-Free Water | Thermo Fisher Scientific | AM9937 | |
Nuclei Buffer (20X) | 10X Genomics | 2000153 | |
Protector RNase inhibitor | Sigma | 3335399001 | |
Sodium chloride (NaCl) | Sigma | S7653-250G | |
Sodium Chloride Solution, 5 M | Sigma | 59222C | |
Trizma Hydrochloride Soultion, pH 7.4 | Sigma | T2194 | |
Trizma hydrochloride (Tris-HCl) (pH7.4) | Sigma | T3252-500G | |
Tween 20 | Bio-Rad | 1662404 | |
Other equipment | |||
Automated cell counter | CytoSMART | 699910591 | |
Microscope | Zeiss | Primostar 3 |
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