The authors thank Jason Hatakeyama (10x Genomics) and Diana Slater (Children&#39;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).

1. Cryopreservation of cells
<ol>
	<li>Freeze &#62;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&#176;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.</li>
	<li>Move from -80 &#176;C to liquid nitrogen storage within 24 h.</li>
</ol>
2. Thawing of cells
<ol>
	<li>Retrieve cryovial from liquid nitrogen storage and thaw in a 37 &#176;C water bath for 2 min. Remove from the water bath while small ice crystals are still visible.</li>
	<li>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 &#176;C.</li>
	<li>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 &#181;L micropipette. Transfer to a 5 mL polystyrene round bottom tube.</li>
</ol>
3. Removal of dead cells
<ol>
	<li>Add 50 &#181;L of dead cell removal cocktail (Table of Materials) to 1 mL of cell suspension. Add 50 &#181;L 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.</li>
	<li>Vigorously vortex for 30 s, the dextran beads designed for the depletion of unwanted cell types labeled with biotin (Table of Materials). Add 100 &#181;L of dextran beads to cell suspension and mix by gently pipetting up and down 2x with a 1000 &#181;L micropipette (Table of Materials).</li>
	<li>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 &#181;L micropipette. Place the tube into the magnet (Table of Materials) and incubate at room temperature for 3 min.</li>
	<li>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&#176;C. Remove most of the supernatant and resuspend in 1 mL of DPBS + 0.04% BSA. Gently mix by pipetting 3x with a 1000 &#181;L micropipette.</li>
</ol>
4. Isolation of nuclei
<ol>
	<li>Centrifuge live cell suspension at 460 x g for 5 min at 4 &#176;C and then aspirate supernatant, ensuring no disruption of the cell pellet.</li>
	<li>Add 100 &#181;L of freshly prepared 0.5x lysis buffer chilled on ice (Table 1) and gently mix by pipetting 10x with a 100 &#181;L micropipette. Incubate 3 min on ice.</li>
	<li>Add 500 &#181;L of chilled wash buffer (Table 2) to the tube without mixing. Centrifuge at 600 x g for 5 min at 4 &#176;C.</li>
</ol>
5. Washing and assessment of nuclei
<ol>
	<li>Aspirate supernatant and add 500 &#181;L of chilled wash buffer to dissolve the remaining intact pellet without mixing. Centrifuge at 600 x g for 5 min at 4 &#176;C. Remove all supernatant without disrupting the nuclei pellet.</li>
	<li>Resuspend in 120 &#181;L of chilled diluted nuclei buffer (Table 3). Filter the cell suspension using a 40 &#181;m cell strainer (Table of Materials).</li>
	<li>Stain with 0.4% trypan blue and visually assess the quality of isolated nuclei under at 10x microscope (Table of Materials).</li>
</ol>
6. Processing isolated nuclei
<ol>
	<li>Keep nuclei on ice. Proceed to further processing immediately, as nuclear clumping can occur over time and necessitate additional filtration steps.</li>
</ol>

High-Quality Nuclei from iPSC-Derived Hematopoietic Cells for Multiomics Studies

<ol>
	<li>Ito, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turbulence+activates+platelet+biogenesis+to+enable+clinical+scale+ex+vivo+production.">Turbulence activates platelet biogenesis to enable clinical scale ex vivo production.</a> Cell. 174 (3), 636-648 (2018).</li><li>An, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+pluripotent+stem+cells+to+generate+diagnostic+tools+for+transfusion+medicine.">The use of pluripotent stem cells to generate diagnostic tools for transfusion medicine.</a> Blood. 140 (15), 1723-1734 (2022).</li><li>Zeng, J., Tang, S. Y., Toh, L. L., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+&amp;#34;Off-the-Shelf&amp;#34;+Natural+Killer+Cells+from+Peripheral+Blood+Cell-Derived+Induced+Pluripotent+Stem+Cells.">Generation of &amp;#34;Off-the-Shelf&amp;#34; Natural Killer Cells from Peripheral Blood Cell-Derived Induced Pluripotent Stem Cells.</a> Stem Cell Rep. 9 (6), 1796-1812 (2017).</li><li>Pavani, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+primitive+and+definitive+erythropoiesis+with+induced+pluripotent+stem+cells.">Modeling primitive and definitive erythropoiesis with induced pluripotent stem cells.</a> Blood Adv. , (2024).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+epigenomic+and+transcriptomic+analysis+reveals+the+requirement+of+JUNB+for+hematopoietic+fate+induction.">Integrative epigenomic and transcriptomic analysis reveals the requirement of JUNB for hematopoietic fate induction.</a> Nat Comm. 13 (1), 3131 (2022).</li><li>Yu, X., Abbas-Aghababazadeh, F., Chen, Y. A., Fridley, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+and+bioinformatics+analysis+of+data+from+bulk+and+single-cell+RNA+sequencing+experiments.">Statistical and bioinformatics analysis of data from bulk and single-cell RNA sequencing experiments.</a> Methods Mol Biol. 2194, 143-175 (2021).</li><li>Jovic, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA+sequencing+technologies+and+applications:+A+brief+overview.">Single-cell RNA sequencing technologies and applications: A brief overview.</a> Clin Transl Med. 12 (3), e694 (2022).</li><li>Choi, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+hemogenic+endothelial+progenitor+and+its+direct+precursor+in+human+pluripotent+stem+cell+differentiation+cultures.">Identification of the hemogenic endothelial progenitor and its direct precursor in human pluripotent stem cell differentiation cultures.</a> Cell Rep. 2 (3), 553-567 (2012).</li><li>Hao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+analysis+of+multimodal+single-cell+data.">Integrated analysis of multimodal single-cell data.</a> Cell. 184 (13), 3573-3587 (2021).</li><li>Slyper, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-cell+and+single-nucleus+RNA-Seq+toolbox+for+fresh+and+frozen+human+tumors.">A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors.</a> Nat med. 26 (5), 792-802 (2020).</li><li>Wu, H., Kirita, Y., Donnelly, E. L., Humphreys, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+of+single-nucleus+over+single-cell+RNA+sequencing+of+adult+kidney:+Rare+cell+types+and+novel+cell+states+revealed+in+fibrosis.">Advantages of single-nucleus over single-cell RNA sequencing of adult kidney: Rare cell types and novel cell states revealed in fibrosis.</a> J Am Soc Nephrol. 30 (1), 23-32 (2019).</li><li>Nadelmann, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+nuclei+from+mammalian+cells+and+tissues+for+single-nucleus+molecular+profiling.">Isolation of nuclei from mammalian cells and tissues for single-nucleus molecular profiling.</a> Curr Protoc. 1 (5), e132 (2021).</li><li>Del-Aguila, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-nuclei+RNA+sequencing+study+of+Mendelian+and+sporadic+AD+in+the+human+brain.">A single-nuclei RNA sequencing study of Mendelian and sporadic AD in the human brain.</a> Alzheimer's Res Ther. 11 (1), 71 (2019).</li><li>Bakken, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-nucleus+and+single-cell+transcriptomes+compared+in+matched+cortical+cell+types.">Single-nucleus and single-cell transcriptomes compared in matched cortical cell types.</a> PloS one. 13 (12), e0209648 (2018).</li><li>Kim, N., Kang, H., Jo, A., Yoo, S. A., Lee, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+single-nucleus+RNA+sequencing+in+different+cell+types+and+tissues.">Perspectives on single-nucleus RNA sequencing in different cell types and tissues.</a> J Pathol Transl Med. 57 (1), 52-59 (2023).</li><li>Maciejowski, J., Hatch, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+membrane+rupture+and+its+consequences.">Nuclear membrane rupture and its consequences.</a> Ann Rev Cell Dev Biol. 36, 85-114 (2020).</li><li>Fang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+analysis+of+single+cell+ATAC-seq+data+with+SnapATAC.">Comprehensive analysis of single cell ATAC-seq data with SnapATAC.</a> Nat Comm. 12 (1), 1337 (2021).</li><li>Baysoy, A., Bai, Z., Satija, R., Fan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+technological+landscape+and+applications+of+single-cell+multi-omics.">The technological landscape and applications of single-cell multi-omics.</a> Nat Rev. Mol Cell Biol. 24 (10), 695-713 (2023).</li></ol>

The authors have no conflict of interest to disclose.

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 ...

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 &#181;m 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.

<table><tbody><tr><td>&lt;strong&gt;Cell Culture Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dimethyl sulfoxide solution (DMSO)</td><td>Sigma</td><td>673439</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline (DPBS)</td><td>Corning</td><td>21031CV</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>GeminiBio</td><td>100106</td><td></td></tr><tr><td>RPMI Medium 1640 (1X)</td><td>Gibco</td><td>11875093</td><td></td></tr><tr><td>&lt;strong&gt;Cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Induced pluripotent stem cells</td><td>N/A</td><td>N/A</td><td>The iPSCs used in this study were obtained through the CHOP Human Pluripotent Stem Cell Core Facility</td></tr><tr><td>&lt;strong&gt;Cell Staining Reagents&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Trypan Blue Solution</td><td>Corning</td><td>25900CI</td><td></td></tr><tr><td>&lt;strong&gt;Dead Cell Removal Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma</td><td>C4901</td><td></td></tr><tr><td>EasySep Dead Cell Removal (Annexin V) Kit</td><td>StemCell Technologies</td><td>17899</td><td>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.</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10 &amp;micro;l Micropipette</td><td>Wards science</td><td>470231606</td><td></td></tr><tr><td>100 &amp;micro;l Micropipette</td><td>Wards science</td><td>470231598</td><td></td></tr><tr><td>1000 &amp;micro;l Extended Universal Tip</td><td>Oxford Lab Products</td><td>OAR-1000XL-SLF</td><td></td></tr><tr><td>1000 &amp;micro;l Micropipette</td><td>Wards science</td><td>470231602</td><td></td></tr><tr><td>2.0 ml Cryogenic vials</td><td>Corning</td><td>431386</td><td></td></tr><tr><td>20 &amp;micro;l Micropipette</td><td>Wards science</td><td>470231608</td><td></td></tr><tr><td>200 &amp;micro;l Micropipette</td><td>Wards science</td><td>470231600</td><td></td></tr><tr><td>5ml Polystyrene Round-Bottom Tube</td><td>Falcon</td><td>352063</td><td></td></tr><tr><td>Corning DeckWork 0.1-10 &amp;micro;l Pipet Tip Station</td><td>Corning</td><td>4143</td><td></td></tr><tr><td>Corning DeckWork 1-200 &amp;micro;l Pipet Tip Station</td><td>Corning</td><td>4144</td><td></td></tr><tr><td>Counting chamber</td><td>CytoSMART</td><td>699910591</td><td></td></tr><tr><td>Flowmi Cell Strainer, 40 &amp;micro;m&amp;nbsp;</td><td>Bel-Art</td><td>H136800040</td><td></td></tr><tr><td>Magnet</td><td>StemCell Technologies</td><td>18000</td><td></td></tr><tr><td>NALGENE Cryo 1&amp;deg;C Freezing Container</td><td>Nalgene</td><td>51000001</td><td></td></tr><tr><td>&lt;strong&gt;Nuclei Isolation Reagents&lt;/strong&gt;</td><td></td><td></td><td>Nuclei isolation reagents include Lysis Buffer,&amp;nbsp; 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).</td></tr><tr><td>Dead Cell Removal kit</td><td>STEMCELL</td><td>17899</td><td></td></tr><tr><td>Digitonin</td><td>Thermo Fisher Scientific</td><td>BN2006</td><td></td></tr><tr><td>DL-Dithiothreitol solution (DTT)</td><td>Sigma</td><td>646563</td><td></td></tr><tr><td>Flowmi Cell Strainer, 40 &amp;micro;m</td><td>Bel-Art</td><td>H136800040</td><td></td></tr><tr><td>MACS bovine serum albumin (BSA) stock Solution</td><td>Miltenyi Biotec</td><td>130091376</td><td></td></tr><tr><td>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma</td><td>7786303</td><td></td></tr><tr><td>Magnesium Chloride Solution, 1 M</td><td>Sigma</td><td>M1028</td><td></td></tr><tr><td>Nonidet P40 Substitute</td><td>Sigma</td><td>74385</td><td></td></tr><tr><td>Nuclease-Free Water</td><td>Thermo Fisher Scientific</td><td>AM9937</td><td></td></tr><tr><td>Nuclei Buffer (20X)</td><td>10X Genomics</td><td>2000153</td><td></td></tr><tr><td>Protector RNase inhibitor</td><td>Sigma</td><td>3335399001</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma</td><td>S7653-250G</td><td></td></tr><tr><td>Sodium Chloride Solution, 5 M</td><td>Sigma</td><td>59222C</td><td></td></tr><tr><td>Trizma Hydrochloride Soultion, pH 7.4</td><td>Sigma</td><td>T2194</td><td></td></tr><tr><td>Trizma hydrochloride (Tris-HCl) (pH7.4)</td><td>Sigma</td><td>T3252-500G</td><td></td></tr><tr><td>Tween 20</td><td>Bio-Rad</td><td>1662404</td><td></td></tr><tr><td>&lt;strong&gt;Other equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Automated cell counter</td><td>CytoSMART</td><td>699910591</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Primostar 3</td><td></td></tr></tbody></table>

nuclear isolation from cryopreserved in vitro derived blood cells

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.

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...

Read this Scientific Article Protocol about Author Spotlight: Advancing In Vitro Blood Cell Production with Single-Cell Multiomics and Functional Genomics at JoVE.com

Author Spotlight: Advancing In Vitro Blood Cell Production with Single-Cell Multiomics and Functional Genomics

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.

Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells

Induced pluripotent stem cell (iPSC)-based models are excellent platforms to understand blood development, and iPSC-derived blood cells have translational ...

nuclear-isolation-from-cryopreserved-in-vitro-derived-blood-cells

Research

JoVE Journal

Developmental Biology

1.0K Views.  Children’s Hospital of Philadelphia. Mijn lab bestudeert genen en mechanismen die de vorming van bloedcellen beïnvloeden. We produceren bloedcellen uit door mensen geïnduceerde pluripotente stamcellen, en we willen dit efficiënter doen om de productie op klinische schaal van celtherapieën te ondersteunen die zijn afgestemd op specifieke patiënten. We gebruiken single-cell genomics-modaliteiten om patronen in genexpressie en andere factoren te identificeren die de vorming van bloedcellen kunnen ondersteunen.Een grote ...

Video: Auteur in de schijnwerpers: In vitro bloedcelproductie bevorderen met single-cell multiomics en functionele genomica

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Medicine

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Immunology and Infection

Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused on bulk approaches that led to the molecular classification of brain tumors. Over the last five years, single cell sequencing approaches have highlighted several important features of gliomas. The majority of these studies have utilized fresh brain tumor specimens to isolate single cells using flow cytometry or antibody-based separation methods. Moving forward, the use of fresh-frozen tissue samples from biobanks will provide greater flexibility to single cell applications. Furthermore, as the single-cell field advances, the next challenge will be to generate multi-omics data from either a single cell or the same sample preparation to better unravel tumor complexity. Therefore, simple and flexible protocols that allow data generation for various methods such as single-nucleus RNA sequencing (snRNA-seq) and single nucleus Assay for Transposase-Accessible Chromatin with high-throughput sequencing (snATAC-seq) will be important for the field.
Recent advances in the single cell field coupled with accessible microfluidic instruments such as the 10x genomics platform have facilitated single cell applications in research laboratories. To study brain tumor heterogeneity, we developed an enhanced protocol for the isolation of single nuclei from fresh frozen gliomas. This protocol merges existing single cell protocols and combines a homogenization step followed by filtration and buffer mediated gradient centrifugation. The resulting samples are pure single nuclei suspensions that can be used to generate single nucleus gene expression and chromatin accessibility data from the same nuclei preparation.

Intra-tumoral heterogeneity is an inherent feature of tumors, including gliomas. We developed a simple and efficient protocol that utilizes a combination of buffers and gradient centrifugation to isolate single nuclei from fresh frozen glioma tissues for single nucleus RNA and ATAC sequencing studies.

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused ...

Cancer Research

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

Examining Micronuclei Yields in Irradiated Whole Blood Samples

The Micronucleus Assay on Cryopreserved Whole Blood

The in vitro cytokinesis-block micronucleus (CBMN) assay is a widely used technique in radiobiology research, biological dosimetry, genotoxicity studies, and in vitro radiosensitivity testing. This cytogenetic method is based on the detection of micronuclei in binucleated cells resulting from chromosomal fragments lagging during cell division. Fresh whole blood samples are the most preferred sample type for the CBMN assay. However, the disadvantages of working with fresh blood samples include immediate processing after blood collection and the limited number of repeated analyses that can be performed without extra blood sampling. 
As the need for fresh blood samples can be logistically challenging, CBMN assay on cryopreserved whole blood samples would be of great advantage, especially in large-scale patient studies. This paper describes a protocol to freeze whole blood samples and to perform the CBMN assay on these frozen blood samples. Blood samples from healthy volunteers have been frozen and thawed at different time points and then, subjected to a modified micronucleus assay protocol. The results demonstrate that this optimized procedure allows the performance of the CBMN assay on frozen blood samples. The described cryopreservation protocol may also be very useful for other cytogenetic assays and a variety of functional assays requiring proliferating lymphocytes.

Author Spotlight: Cryopreservation of Whole Blood Samples for High Throughput Analysis 

Here we present an optimized protocol for the cytokinesis-block micronucleus assay on cryopreserved whole blood samples. This optimized method of cryopreservation of whole blood for micronucleus analysis is a reliable technique for large-scale sampling and multi-center studies and can be used for other blood-related assays as well.

The in vitro cytokinesis-block micronucleus (CBMN) assay is a widely used technique in radiobiology research, biological dosimetry, genotoxicity studies, ...

Nuclei Isolation from Mouse Cardiac Progenitor Cells at Single-Cell Resolution

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

Author Spotlight: Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution  

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

PBMC Collection and Cryostorage for Mitochondrial Bioenergetics Analysis

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

Author Spotlight: Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...

Isolation and Cryopreservation of Highly Viable Human Peripheral Blood Mononuclear Cells From Whole Blood: A Guide for Beginners

Peripheral blood mononuclear cells (PBMCs) are commonly used in biomedical research on the immune system and its response to disease and pathogens. This detailed protocol describes the equipment, supplies, and steps for isolating, cryopreserving, and thawing high-quality and highly viable PBMCs from whole blood cells suitable for downstream applications such as flow cytometry and RNA-sequencing. Protocols for processing plasma and buffy coat from the whole blood in parallel and concurrently with PBMCs are also described. This easy-to-follow step-by-step protocol, which utilizes density gradient centrifugation to isolate PBMCs, is accompanied by a checklist of supplies, equipment, and preparation steps. This protocol is suitable for individuals with any prior experience with laboratory techniques and can be implemented in clinical or research laboratories. High-quality cell viability and RNA sequencing resulted from PBMCs collected by operators with no prior laboratory experience using this protocol.

This protocol presents an accessible guide for collecting, storing, and thawing peripheral blood mononuclear cells suitable for downstream analyses and workflows like flow cytometry and RNA sequencing. Plasma and buffy coat collections are also demonstrated.

Peripheral blood mononuclear cells (PBMCs) are commonly used in biomedical research on the immune system and its response to disease and pathogens. This ...

Isolation of Nuclei from Flash-Frozen Liver Tissue for Single-Cell Multiomics

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy homeostasis and the metabolism of xenobiotics, among others. These tasks are performed through tight coordination between hepatic parenchymal and non-parenchymal cells. Additionally, various metabolic activities are confined to specific areas of the hepatic lobule-a phenomenon called liver zonation. Recent advances in single-cell sequencing technologies have empowered researchers to investigate tissue heterogeneity at a single-cell resolution. In many complex tissues, including the liver, harsh enzymatic and/or mechanical dissociation protocols can negatively affect the viability or the quality of the single-cell suspensions needed to comprehensively characterize this organ in health and disease.
This paper describes a robust and reproducible protocol for isolating nuclei from frozen, archived liver tissues. This method yields high-quality nuclei that are compatible with downstream, single-cell omics approaches, including single-nucleus RNA-seq, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), as well as multimodal omics (joint RNA-seq and ATAC-seq). This method has been successfully used for the isolation of nuclei from healthy and diseased human, mouse, and non-human primate frozen liver samples. This approach allows the unbiased isolation of all the major cell types in the liver and, therefore, offers a robust methodology for studying the liver at the single-cell resolution.

Here, we present a protocol for isolating nuclei from flash-frozen, archived liver tissues for single-nucleus RNA-seq, ATAC-seq, and joint multiomics (RNA-seq and ATAC-seq).

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy ...

Biology

Isolation and Profiling of Single Nuclei from Frozen Mammalian Tissues

A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.

Author Spotlight: Enhancing Drug Discovery - Development of Automated, Standardized Protocols for Nuclei Extraction from Frozen Tissues 

The study describes a simple, quick, and partially automated protocol to isolate high-quality nuclei from frozen mammalian tissues for downstream single nuclei RNA sequencing.

Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells

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