Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells
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 have been used extensively to investigate iPSC-based developmental biology, including cell types that are produced during hematopoiesis5. Compared to bulk RNA sequencing, which cannot infer relationships between cell subpopulations6, single-cell modalities allow for assessments of cell heterogeneity and can facilitate the identification and characterization of cell development6,7.
The formation of hematopoietic cells from iPSCs requires sequential differentiation through primitive streak, mesoderm, and endothelial states to form hemogenic endothelial cells. Hemogenic endothelial cells are direct precursors to hematopoietic stem and progenitor cells8. These cell types have remarkably different morphological and cellular properties. There is a limited understanding of the epigenetic landscape and developmental dynamics of in vitro-derived hematopoietic cell models, though this is essential knowledge to unlock the full therapeutic potential of the iPSC system. In many cases, only single cell analysis modalities allow for the identification of cell populations, transcriptional activities, chromatin landscapes, and regulatory mechanisms that govern development among heterogenous cell preparations.
Two methodologies, single-cell RNA sequencing (scRNAseq) and single nuclei RNA sequencing (snRNAseq), can reveal transcriptional insights at the individual cell level9. A principal factor dictating data validity and reliability is the uncompromising need for samples that meet stringent quality criteria. These include precise requirements for cell/nuclear density, optimal viability, and minimal clumping. Preparation of single nuclei can be technically challenging. There can be additional challenges associated with the use of previously cryopreserved cells.
We note four important potential limitations to standard scRNAseq approaches. First, standard protocols for generating cell suspensions often reference preparations from fresh cells or tissues, rather than those retrieved post-cryopreservation10. This can curtail the utility of potentially valuable resources. Second, the dissociation efficiency of diverse cell types is variable. For instance, many immune cell types undergo dissociation with relative ease. In contrast, stromal cells, fibroblasts, and endothelial cells, which are normally embedded in the extracellular matrix and basement membrane, have unique cell properties which can complicate nuclei isolation11,12. These properties can necessitate aggressive dissociation protocols, which may jeopardize the structural integrity of more delicate cell populations. Third, some cells (e.g., megakaryocytes) can surpass 100 µm in diameter and pose difficulties for several existing commercial single-cell platforms13. Additionally, improper handling can lead to cellular damage and stress responses during the initial steps of cell isolation, involving enzymatic hydrolysis and mechanical dissociation, which can impact gene expression profiles11,14.
For these and other reasons, a single nucleus approach may be a preferable alternative to single cell methods15. Given the superior stability of the nuclear membrane compared to the cell membrane, the nuclear envelope may remain intact even after tissue cryopreservation16. This can facilitate nuclear extraction and enhance the diversity of sample types suitable for next generation sequencing modalities. Second, nuclei exhibit heightened resistance to mechanical perturbations and tend to manifest fewer transcriptional shifts during dissociation14. Therefore, nuclei can provide greater RNA stability and more reproducible transcriptional profiles. A third noteworthy advantage of single nuclei methods is a lack of cell size constraints and an applicability for larger cells, like cardiomyocytes or megakaryocytes12. Fourth, nuclei serve as suitable substrates for multiomics analyses, which offer expanded insights from integrated analysis of gene expression, accessible chromatin regions, and other parameters17, enabling deeper understanding of cell heterogeneity, development, and functional states18.
By profiling iPSCs during hematopoietic development, multiomics approaches can help define developmental processes in normal or pathologic disease models. Comparisons of iPSC-derived cells to primary cells or tissues can also provide an assessment of iPSC model fidelity to in vivo biology, facilitating iPSC model improvement and the discovery of novel cell populations and factors driving blood formation.
Although many groups have successfully navigated nuclear isolation and resultant next generation sequencing analysis, isolating high quality nuclei remains a barrier to some laboratories interested in performing single nucleus-based analyses. This manuscript details a protocol for isolating quality nuclei from previously cryopreserved iPSC-derived cells. We have focused on adherent stromal/endothelial cells and non-adherent hematopoietic progenitor cells, as these represent very different cell types with regard to structure and content that demand tailored modifications and troubleshooting to heighten recovery of high-quality nuclei amenable for next generation sequencing or other experiments.
Protocol
1. Cryopreservation of cells
- 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.
- Move from -80 °C to liquid nitrogen storage within 24 h.
2. Thawing of cells
- Retrieve cryovial from liquid nitrogen storage and thaw in a 37 °C water bath for 2 min. Remove from the water bath while small ice crystals are still visible.
- Transfer the cells from the cryovial to an empty 15 mL tube and slowly add 10 mL of pre-warmed medium (RPMI 1640 + 10% FBS) while mixing carefully. Centrifuge at 400 x g for 3 min at 25 °C.
- Aspirate the supernatant and reconstitute in 1 mL of DPBS supplemented with 2% FBS and 1 mM CaCl2 (Table of Materials). Carefully mix by pipetting 3x with a 1000 µL micropipette. Transfer to a 5 mL polystyrene round bottom tube.
3. Removal of dead cells
- Add 50 µL of dead cell removal cocktail (Table of Materials) to 1 mL of cell suspension. Add 50 µL of Biotin selection cocktail to the cell mixture. Incubate at room temperature for 3 min. These steps label Annexin V+ dead cells contained within the cell suspension with biotin.
- Vigorously vortex for 30 s, the dextran beads designed for the depletion of unwanted cell types labeled with biotin (Table of Materials). Add 100 µL of dextran beads to cell suspension and mix by gently pipetting up and down 2x with a 1000 µL micropipette (Table of Materials).
- Add 1.3 mL of DPBS containing 2% FBS and 1 mM CaCl2 to the cell suspension and mix by gently pipetting up and down 2x with a 1000 µL micropipette. Place the tube into the magnet (Table of Materials) and incubate at room temperature for 3 min.
- Invert the magnet and tube to pour the enriched live cell suspension into a new 15 mL conical tube. Centrifuge at 400 x g for 3 min at 4°C. Remove most of the supernatant and resuspend in 1 mL of DPBS + 0.04% BSA. Gently mix by pipetting 3x with a 1000 µL micropipette.
4. Isolation of nuclei
- Centrifuge live cell suspension at 460 x g for 5 min at 4 °C and then aspirate supernatant, ensuring no disruption of the cell pellet.
- Add 100 µL of freshly prepared 0.5x lysis buffer chilled on ice (Table 1) and gently mix by pipetting 10x with a 100 µL micropipette. Incubate 3 min on ice.
- Add 500 µL of chilled wash buffer (Table 2) to the tube without mixing. Centrifuge at 600 x g for 5 min at 4 °C.
5. Washing and assessment of nuclei
- Aspirate supernatant and add 500 µL of chilled wash buffer to dissolve the remaining intact pellet without mixing. Centrifuge at 600 x g for 5 min at 4 °C. Remove all supernatant without disrupting the nuclei pellet.
- Resuspend in 120 µL of chilled diluted nuclei buffer (Table 3). Filter the cell suspension using a 40 µm cell strainer (Table of Materials).
- Stain with 0.4% trypan blue and visually assess the quality of isolated nuclei under at 10x microscope (Table of Materials).
6. Processing isolated nuclei
- Keep nuclei on ice. Proceed to further processing immediately, as nuclear clumping can occur over time and necessitate additional filtration steps.
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 processing, as debris or clumped nuclei can diminish or prevent the generation of quality data due to clogs in precision microfluidic instruments. Microscopic examination of nuclei isolated from a sample that was prepared using a conventional kit protocol showed the presence of debris and ≥1 nuclear aggregate per visualized field (Figure 2A-B). These samples were discarded due to inferior quality.
High quality nuclei are round and intact, with minimal to no clumps, such as those that we observed after method optimization (Figure 2C-D). We observed no aggregates or clumping in these samples. These nuclei were of sufficient quality and quantity to support downstream processing and analysis. Similar troubleshooting and protocol optimization can potentially extend to other cryopreserved cell types.
Figure 1: Diagram illustrating nuclei extraction from cryopreserved cells. Visual depiction detailing steps involved in nuclear isolation from cryopreserved cells. Please click here to view a larger version of this figure.
Figure 2: Nuclei isolated from cryopreserved human iPSC-derived cells. Isolated nuclei from adherent stromal/endothelial cells stained with trypan blue show significant debris (A) small particles and (B) nuclear aggregates (large particle) that precluded downstream analyses. After performing technical modifications presented here, high quality nuclei were isolated from (C) cryopreserved human iPSC-derived hematopoietic progenitor cells or (D) stromal/endothelial cell populations in sufficient quantities to support downstream analyses. For reference, these non-adherent hematopoietic progenitor cells were collected at day 9 + 8 and adherent stromal/endothelial cells on day 9 + 7 of hematopoietic differentiation as described in4. Note that nuclear morphology can differ across cell types. Scale bar: 100 µm. Please click here to view a larger version of this figure.
| Buffer Preparation | |||
| 0.5X Lysis buffer | Stock | Final | 2 mL |
| 1X Lysis Buffer | 1X | 0.5X | 1 mL |
| Lysis Dilution Buffer | 1 mL | ||
| Lysis Buffer | Stock | Final | 2 mL |
| BSA | 10% | 1% | 200 µL |
| Digitonin | 5% | 0.01% | 4 µL |
| DTT | 1000 mM | 1 mM | 2 µL |
| MgCl2 | 1 M | 3 mM | 6 µl |
| NaCl | 5 M | 10 mM | 4 µL |
| Nonidet P40 Substitute | 10% | 0.10% | 20 µL |
| Nuclease-free Water | 1.67 mL | ||
| RNase inhibitor | 40 U/µl | 1 U/µl | 50 µl |
| Tris-HCl (pH7.4) | 1 M | 10 mM | 20 µL |
| Tween-20 | 10% | 0.10% | 20 µL |
| Lysis Dilution Buffer | Stock | Final | 2 Ml |
| BSA | 10% | 1% | 200 µl |
| DTT | 1000 mM | 1 mM | 2 µL |
| MgCl2 | 1 M | 3 mM | 6 µL |
| NaCl | 5 M | 10 mM | 4 µL |
| Nuclease-free Water | 1.72 mL | ||
| RNase inhibitor | 40 U/µl | 1 U/µl | 50 µl |
| Tris-HCl (pH7.4) | 1 M | 10 mM | 20 µl |
Table 1: Preparation of 0.5x Lysis buffer. This solution is designated for the nuclei isolation procedure in step 4.2. Lysis buffer and lysis dilution buffer are mixed to prepare 0.5x lysis buffer. It is imperative to prepare fresh buffer immediately prior to each experiment and to keep on ice. Note that troubleshooting for some cell types may involve 1x, 0.5x, or lower dilutions of lysis buffer to facilitate intact nucleus isolation.
| Buffer Preparation | |||
| Wash Buffer | Stock | Final | 4 mL |
| BSA | 10% | 1% | 400 µL |
| DTT | 1000 mM | 1 mM | 4 µL |
| MgCl2 | 1 M | 3 mM | 12 µL |
| NaCl | 5 M | 10 mM | 8 µL |
| Nuclease-free Water | 3.40 mL | ||
| RNase inhibitor | 40 U/µl | 1 U/µl | 100 µL |
| Tris-HCl (pH7.4) | 1 M | 10 mM | 40 µL |
| Tween-20 | 10% | 0.10% | 40 µL |
Table 2: Preparation of wash buffer. This solution is utilized during nuclei isolation. This should be prepared fresh prior to each experiment and kept on ice.
| Buffer Preparation | |||
| Diluted Nuclei Buffer | Stock | Final | 1 mL |
| DTT | 1000 mM | 1 mM | 1 µL |
| Nuclease-free Water | 924 µL | ||
| Nuclei Buffer | 20X | 1X | 50 µL |
| RNase inhibitor | 40 U/µL | 1 U/µL | 25 µL |
Table 3: Preparation of diluted nuclei buffer. This solution is used in step 5.2. It should be prepared fresh prior to each experiment and kept on ice.
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 samples and for rare iPSC-derived cell populations.
It is important to tailor lysis buffer dilution, lysis time, and filtration frequency for specific cell populations. We have observed particularly sensitive cell types, such as cryopreserved human iPSC-derived hematopoietic progenitor cells, to become over-lysed in less than a minute with 1x lysis buffer. Such over-lysis is not merely a technical or operational malfunction. Rather, over-lysis and clumping can indicate leakage of adhesive nuclear materials. For adherent stromal and endothelial cells with robust cytoskeletal architecture and membranes with a propensity to form strong cell-cell adhesions, double filtration was necessary to remove clumped nuclei. Nuclear aggregates can obstruct microfluidic channels, presenting tangible risks to experimental integrity and potentially precluding any data output.
We also found that incorporating a dead cell removal step dramatically increased recovery of intact nuclei while limiting clumping and debris. It has been necessary to process samples containing >70% viable cells based on trypan blue staining at the start of the experiment. Keeping all buffers on ice can also help maintain viability.
Limitations of the technique
The described method was designed to optimize nuclear recovery from cultured iPSC-derived cell types4. It is possible that similar troubleshooting procedures would extend beyond iPSC-induced cells to include other cryopreserved cell types, but it is somewhat difficult to predict specific needs for individual samples or cell types without testing. Regardless of sample or cell type, aiming for nuclear preparations with <5% remaining viable whole cells, minimizing aggregation, and reducing over-lysed nuclei should directly correlate with experimental success.
Significance with respect to existing methods
Translationally relevant iPSC-based differentiation systems can produce a multitude of cell types and progenitors. This protocol focuses on iPSC-derived adherent stromal/endothelial cells and non-adherent hematopoietic progenitor cells as exemplary cell types that had different methodologic requirements to optimize nuclear isolation. We anticipate that this protocol can be adapted and applied to other iPSC-derived cell types to interrogate pivotal developmental stages, recognizing that it is necessary to fine-tune isolation methods depending on protein content, membrane structure, and cell rigidity. However, the methodology described here may permit recovery of high-quality nuclei from a broad spectrum of cryopreserved cell types.
Divulgaciones
The authors have nothing to disclose.
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
| 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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