Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 reagents^1,2,3,4. 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 development^6,7.

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 isolation^11,12. 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 profiles^11,14.

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.

Protokół

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.

2. Thawing of cells

1. 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.
2. 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.
3. 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

1. 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.
2. 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).
3. 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.
4. 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

1. 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.
2. 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.
3. 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

1. 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.
2. Resuspend in 120 µL of chilled diluted nuclei buffer (Table 3). Filter the cell suspension using a 40 µm cell strainer (Table of Materials).
3. 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

1. Keep nuclei on ice. Proceed to further processing immediately, as nuclear clumping can occur over time and necessitate additional filtration steps.

Wyniki

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...

Dyskusje

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 ...

Materiały

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