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.

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 ...

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Developmental Biology

972 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. 

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

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and mammalian final host, respectively. The ability to isolate and sustain the different life cycle stages under in vitro culture conditions has greatly facilitated investigations of the cellular, biochemical and molecular mechanisms regulating parasite growth, development and host interactions. Transmission of schistosomiasis requires asexual reproduction and development of multiple larval stages within the snail host; from the infective miracidium, through primary and secondary sporocysts, to the final cercarial stage that is infective to humans. In this paper we present a step-by-step protocol for mass hatching and isolation of Schistosoma mansoni miracidia from eggs obtained from livers of infected mice, and their subsequent introduction into in vitro culture. It is anticipated that the detailed protocol will encourage new researchers to engage in and broaden this important field of schistosome research.

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

This article describes a protocol for the large-scale axenic isolation and collection of free-swimming miracidia of the human blood fluke Schistosoma mansoni and their subsequent processing for introduction into in vitro culture.

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...