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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell Culture Reagents</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&#39;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>Cells</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>Cell Staining Reagents&#160;</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>Dead Cell Removal Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride (CaCl2)</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>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;l Micropipette</td>
			<td>Wards science</td>
			<td>470231606</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;l Micropipette</td>
			<td>Wards science</td>
			<td>470231598</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;l Extended Universal Tip</td>
			<td>Oxford Lab Products</td>
			<td>OAR-1000XL-SLF</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;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 &#181;l Micropipette</td>
			<td>Wards science</td>
			<td>470231608</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;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 &#181;l Pipet Tip Station</td>
			<td>Corning</td>
			<td>4143</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning DeckWork 1-200 &#181;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 &#181;m&#160;</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&#176;C Freezing Container</td>
			<td>Nalgene</td>
			<td>51000001</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclei Isolation Reagents</td>
			<td></td>
			<td></td>
			<td>Nuclei isolation reagents include Lysis Buffer,&#160; 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 &#181;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 (MgCl2)</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>Other equipment</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.

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

Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells

My lab studies genes and mechanisms that affect blood cell formation. We produce blood cells from human-induced pluripotent stem cells, and we wanna do this more efficiently to support clinical-scale production of cell therapies that are tailored for specific patients. We use single cell genomics modalities to identify patterns in gene expression and other factors that can help support blood cell formation.One major challenge for in vitro blood cell production is that is expensive and inefficient. We are using genetic approaches to address this. We want to use clues from human genetics and in vivo hematopoiesis to guide the improvements that we make to the in vitro system.We're trying to use functional genomics, and single nucleus analyses to advance the field in terms of identifying genes and signaling networks that drive the formation of the right types of blood cells in vitro. Creating high quality multi-omic data set has helped us to discover transcription factors, energy, and regular machinery that drive in vitro differentiation of blood cells. We are using this data to understand human genetic data like loci, and uncertain factors that are driving differentiation that we haven't been able to see before.We're very excited to build on insights from these multimodal data sets that we've been able to generate based on the isolation of high quality nuclei. We intend to focus our future studies on genes and developmental mechanisms that can produce blood cell types, ultimately at clinical scale in vitro.

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

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 ...

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

Research

JoVE Journal

Developmental Biology

Preparation of Enriched Live Cell Suspension for Nuclei Isolation from iPSC-Derived Blood Cells

Isolating Nuclei from iPSC-Derived Cells for Advanced Multiomics Research

805 Views.  Children’s Hospital of Philadelphia.  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. 

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

概要