This work was supported by NYSTEM grant C030136 to R.H. We would like to thank Bartek Jablonski for his feedback and revision of the manuscript

The HSC ex vivo expansion protocol follows the guidelines of the Research Ethics Committee at Mount Sinai School of Medicine.
1. Buffer and Media Preparation
<ol>
	<li>Prepare separation buffer 24 h prior to isolation of CD34+ cells from UBCs by adding 2 mL of 0.5 M Ethylene Diamine Tetra-acetic Acid (EDTA) and 33 mL of 7.5% Bovine Serum Albumin (BSA) to 465 mL buffered saline (1x PBS). Mix gently and maintain the buffer overnight at 4 &#176;C.</li>
	<li>Warm media at room temperature on the day of purification.</li>
</ol>
2. Isolation of Mononuclear Cells (MNCs) from UCB Unit
<ol>
	<li>Dispense the whole UCB unit into a 75 cm2 flask. Dilute and gently mix the UCB with an equal volume of PBS at room temperature.</li>
	<li>Determine the number of tubes required for processing the whole UCB unit (one 50 mL conical tube can be used to process 35 mL of diluted UCB). Add 15 mL of density gradient media (see Table of Materials) to each tube and dispense the diluted UCB on the top of the density gradient media creating two layers. 
	NOTE: Dispense the diluted UCB very slowly while holding the tube at a 45&#176; angle to prevent mixing of the density gradient media layer with the UCB.</li>
	<li>Centrifuge at 400 x g for 30 min at a low acceleration and deceleration rate. After centrifugation, MNCs locate in the white layer (buffy coat) between the plasma and density gradient media layer.</li>
	<li>Slowly aspirate 2/3 of the plasma without disturbing the buffy coat layer. Carefully transfer the buffy coat layer into a new 50 mL tube. Collect all MNCs from the same UCB unit into a 50 mL tube until reaching 25 mL. Use a new tube if the volume of collected MNCs exceeds 25 mL.</li>
	<li>Add 25 mL of cold PBS into the tube containing 25 mL of MNCs and mix well. Centrifuge at 400 x g for 10 min at 4 &#176;C. 
	NOTE: Cold PBS is important to prevent capping of antibodies on the cell surface and reduce non-specific labeling.</li>
	<li>Carefully aspirate the supernatant and gently re-suspend the cell pellet in 50 mL of cold PBS.</li>
	<li>Remove 20 &#956;L of the cell suspension for counting. Count the cell number stained with acridine orange/propidium iodide staining by using an automated cell counter. Calculate the total number of MNCs. 
	NOTE: The cell count can be also performed by the trypan blue exclusion method using a hemocytometer.</li>
	<li>Centrifuge at 400 x g for 15 min at 4 &#176;C. 
	NOTE: If not required immediately, MNCs can be frozen at this stage.</li>
</ol>
3. Isolation of CD34+ Cells from UCBs
<ol>
	<li>Prepare the CD34+ antibody coupled with magnetic beads solution for isolation of CD34+ cells from MNCs. Mix 300 &#181;L of separation buffer with 100 &#181;L of human FcR human IgG (blocking reagent) and 100 &#181;L of CD34 magnetic beads for isolation of CD34+ cells from each 108 MNCs. For isolation of a higher number of CD34+ cells from (&#62;108) MNCs, scale up reagents accordingly.</li>
	<li>Carefully aspirate the supernatant from the tube centrifuged at step 2.8. Resuspend the pellet in the CD34 magnetic beads solution prepared in step 3.1 (use 500 &#181;L of solution per 108 cells).</li>
	<li>Mix gently and incubate at 4 &#176;C for 30 min. 
	NOTE: Prepare the cell separator device (see Table of Materials) during the incubation period. Replace the storage solution with the working buffer as indicated by the manufacturer&#8217;s instructions and run a washing program followed by a rinsing program.</li>
	<li>Add cold cell separator running buffer to the tube containing the cell mixture until the tube is fully filled. Centrifuge at 400 x g for 15 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant and resuspend the cell pellet in cold cell separator running buffer (2 mL/108 cells). Transfer the 2 mL of cell suspension mixture into 15 mL tubes.</li>
	<li>Load 3 sets of 15 mL tubes into the cell separator rack prechilled at 4 &#176;C. One set of tubes contains MNCs, the second set of tubes will be used to collect the negative fraction and the third set of tubes will be used to collect purified CD34+ cells.</li>
	<li>Load the rack into the cell separator device and run the posseld2 preset program. 
	NOTE: An alternative method that utilizes manual magnetic separator can be used to replace the cell separator device12 .</li>
	<li>Centrifuge tubes with the positive fraction at 400 x g for 15 min at 4 &#176;C. Aspirate the supernatant and resuspend purified CD34+ cells in 1 mL of serum-free media.</li>
	<li>Count the cells stained with acridine orange/propidium iodide using an automated cell counter. 
	NOTE: If not required immediately, purified CD34+ cells can be frozen at this stage.</li>
</ol>
4. VPA Ex Vivo Expansion of purified UCB-CD34+ Cells
<ol>
	<li>Prepare sufficient volume of media to plate the purified CD34+ cells at a density of 3.3 x 104 cells/mL. The culture media composition is serum Free culture medium (see Table of Materials) supplemented with 10 &#181;L/mL pen/strep, 150 ng/mL stem cell factor (SCF), 100 ng/mL fms-like tyrosine kinase receptor 3 (FLT3 ligand), 100 ng/mL thrombopoietin (TPO), and 50 ng/mL interleukin 3 (IL-3).</li>
	<li>Plate purified CD34+ cells in a 12-well plate (5 x 104 CD34+ cells/ 1.5 mL of media/ well) and culture at 37 &#176;C in a 5% CO2 humidified incubator.</li>
	<li>Assess the purity of isolated CD34+ cells and cell composition by FACS analysis as described below in step 6.3.</li>
	<li>Add VPA at a final concentration of 1 mM into the cultures of cells treated for 16 h with cytokine cocktail.</li>
	<li>Culture the cells for an additional 7 days at 37 &#176;C in a 5% CO2 humidified incubator. 
	NOTE: Media remains unchanged during the duration of the ex vivo expansion.</li>
</ol>
5. Antibody Staining for Flow Cytometry Analysis
<ol>
	<li>Prepare antibody-staining solution to stain 2 x 104&#8211;6 x 104 cells and isotype solution to determine FACS gating strategy and determine the level of non-specific binding. Dilute antibodies (1/100 APC anti-CD34, 1/200 FITC anti-CD90) and the respective isotypes into 50 &#181;L of separation buffer (composition of separation buffer is defined in step 1.1). 
	NOTE: Perform single staining of cells with each antibody for a FMO gating strategy. Total antibody compensation bead kit (see Table of Materials) can be used for compensation setup.</li>
	<li>Homogenize the cell culture by pipetting multiple times. Count cells and pipet 5 x 104 cells into a 1.5 mL tube.</li>
	<li>Add 1 mL of separation buffer and centrifuge at 400 x g for 15 min at room temperature.</li>
	<li>Aspirate the supernatant and resuspend the pellet in 50 &#181;L of the staining solution. Incubate cells for 30 min at room temperature.</li>
	<li>Add 450 &#181;L of separation buffer and centrifuge at 400 x g for 15 min at room temperature.</li>
	<li>Aspirate the supernatant and resuspend the pellet in 100 &#181;L of separation buffer. Keep cells on ice for at least 5 min until performing FACS analysis. Add 1 &#181;L of 7-AAD to gate viable cells.</li>
	<li>Load the stained cell sample on the FACS machine and acquire cells at the lowest flow rate.</li>
	<li>For each fluorophore, analyze the isotype controls and single stained cells to set up gating for FITC (CD90), APC (CD34), and PerCP/Cy5.5 (7-AAD) staining.</li>
	<li>Gate PerCP/Cy5.5 negative fraction and plot APC vs FITC to determine the percent of viable CD34+, CD90+, and CD34+CD90+ cells that defines the HSPC/HSC population in the culture.</li>
</ol>
6. Antibody Staining for Flow Cytometry Cell Sorting
<ol>
	<li>Resuspend the expanded cells by pipetting multiple times. Count the cell number and transfer the whole culture into a 15 mL or 50 mL tube.</li>
	<li>Fill up the tube with cold separation buffer</li>
	<li>Centrifuge at 400 x g for 15 min at 4 &#176;C.</li>
	<li>Prepare antibody-staining solution to stain 5 x 105&#173;&#8211;2 x 106 cells and isotype solution to stain 1 x 105 cells and determine FACS gating. Dilute 1/10 (APC anti-CD34), 1/20 (FITC-CD90) antibodies and the corresponding isotypes into 500 &#181;L of separation buffer per each condition.</li>
	<li>Prepare the single color compensation controls using a total antibody compensation bead kit according to manufacturer&#39;s instructions.</li>
	<li>Transfer the supernatant from step 5.2 into a new tube and keep it at 37 &#176;C. This culture media will be reused to culture the sorted cells.</li>
	<li>Resuspend the cell pellet in cold separation buffer to obtain a concentration of 5 x 105 cells/ mL. Transfer 1 x 105 cells in 1.5 mL tubes for isotype staining and the remaining cells that will be sorted into other 1.5 mL tubes.</li>
	<li>Centrifuge the tubes at 400 x g for 15 min at 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend pellets of cells in isotype staining solution and the pellet of cells that will be sorted in the antibody staining solution.</li>
	<li>Incubate for 30 min at 4 &#176;C.</li>
	<li>Add 450 &#181;L of separation buffer and centrifuge at 400 x g for 15 min at 4 &#176;C.</li>
	<li>Resuspend the pellets with 2 mL of cold separation buffer.</li>
	<li>To prevent clogs, filter the samples through a 40 &#956;m cell strainer and place on ice until FACS.</li>
	<li>Set-up cell sorter for sorting with the correct parameters.</li>
	<li>Run isotype samples to set voltage and gain for forward and side scatter and identify negative fluorescence populations.</li>
	<li>Run single color controls to set compensation coefficients and apply compensated parameter to collection protocol.</li>
	<li>Run a small aliquot of the (Ab) sample (~50,000 events) to establish the gating strategy.</li>
	<li>Set up sort decisions to collect the CD34+ CD90- cell fraction.</li>
	<li>Sort cells into collection tubes coated with 2 mL separation buffer.</li>
	<li>After sorting, recount the cells and centrifuge them at 400 x g for 15 min at 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the pellet in the media recovered in step 5.5 to reach a final concentration of 3 x 104 cells/mL.</li>
	<li>Plate CD34+ CD90- purified cells in 12-well plates with 1.5 mL medium/well (5 x 104 CD34+ CD90- cells/1.5 mL of media/well).</li>
	<li>Assess the purity by FACS analysis using 50 &#181;L of media containing cells.</li>
	<li>Treat the cultures of CD34+CD90- cells with VPA at 1mM final concentration.</li>
	<li>Incubate at 37 &#176;C in a 5% CO2 humidified incubator.</li>
</ol>

<ol>
	<li>Broxmeyer, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+the+efficacy+of+engraftment+of+cord+blood+for+hematopoietic+cell+transplantation.">Enhancing the efficacy of engraftment of cord blood for hematopoietic cell transplantation.</a> Transfusion and Apheresis Science. 54 (3), 364-372 (2016).</li><li>Boitano, A. 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=Aryl+hydrocarbon+receptor+antagonists+promote+the+expansion+of+human+hematopoietic+stem+cells.">Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells.</a> Science. 329 (5997), 1345-1348 (2010).</li><li>Fares, I., 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=Cord+blood+expansion.+Pyrimidoindole+derivatives+are+agonists+of+human+hematopoietic+stem+cell+self-renewal.">Cord blood expansion. Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal.</a> Science. 345 (6203), 1509-1512 (2014).</li><li>Chaurasia, P., Gajzer, D. C., Schaniel, C., D&#39;Souza, S., Hoffman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+reprogramming+induces+the+expansion+of+cord+blood+stem+cells.">Epigenetic reprogramming induces the expansion of cord blood stem cells.</a> Journal of Clinical Investigation. 124 (6), 2378-2395 (2014).</li><li>Wagner, J. 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=Phase+I/II+Trial+of+StemRegenin-1+Expanded+Umbilical+Cord+Blood+Hematopoietic+Stem+Cells+Supports+Testing+as+a+Stand-Alone+Graft.">Phase I/II Trial of StemRegenin-1 Expanded Umbilical Cord Blood Hematopoietic Stem Cells Supports Testing as a Stand-Alone Graft.</a> Cell Stem Cell. 18 (1), 144-155 (2016).</li><li>Peled, T., 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=Nicotinamide,+a+SIRT1+inhibitor,+inhibits+differentiation+and+facilitates+expansion+of+hematopoietic+progenitor+cells+with+enhanced+bone+marrow+homing+and+engraftment.">Nicotinamide, a SIRT1 inhibitor, inhibits differentiation and facilitates expansion of hematopoietic progenitor cells with enhanced bone marrow homing and engraftment.</a> Experimental Hematology. 40 (4), (2012).</li><li>Nikiforow, S., Ritz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dramatic+Expansion+of+HSCs:+New+Possibilities+for+HSC+Transplants?.">Dramatic Expansion of HSCs: New Possibilities for HSC Transplants?.</a> Cell Stem Cell. 18 (1), 10-12 (2016).</li><li>Mehta, R. S., 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=Novel+Techniques+for+Ex+vivo+Expansion+of+Cord+Blood:+Clinical+Trials.">Novel Techniques for Ex vivo Expansion of Cord Blood: Clinical Trials.</a> Frontiers in Medicine. 2, 89 (2015).</li><li>Papa, 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=Ex+vivo+human+HSC+expansion+requires+coordination+of+cellular+reprogramming+with+mitochondrial+remodeling+and+p53+activation.">Ex vivo human HSC expansion requires coordination of cellular reprogramming with mitochondrial remodeling and p53 activation.</a> Blood Advances. 2 (20), 2766-2779 (2018).</li><li>Papa, L., Djedaini, M., Hoffman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Role+in+Stemness+and+Differentiation+of+Hematopoietic+Stem+Cells.">Mitochondrial Role in Stemness and Differentiation of Hematopoietic Stem Cells.</a> Stem Cells International. , (2019).</li><li>Genovese, P., 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=Targeted+genome+editing+in+human+repopulating+haematopoietic+stem+cells.">Targeted genome editing in human repopulating haematopoietic stem cells.</a> Nature. 510 (7504), 235-240 (2014).</li><li>Perdomo, J., Yan, F., Leung, H. 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The authors have nothing to disclose.

Herein, we present a protocol to rapidly expand to a significant degree the number of functional human HSCs from UCBs. The pilot and kinetic studies using this protocol indicate that the ex vivo expanded cells promptly acquire and retain the expression of several HSC phenotypic markers including CD90 as well as primitive HSC metabolic characteristics9.
This ex vivo expansion protocol is relatively simple and reliable. Purification of CD34+ cells with the cell...

Ex vivo expansion of hematopoietic stem cells (HSCs) from umbilical cord blood (UCB) units holds great promise for HSC applications in regenerative medicine and transplantation therapy. Transplantation with UCB units has several unique advantages such as easy collection, high availability, minimal risk of infection, low risk of disease relapse, and low frequency of graft-versus host disease (GVHD). However, the major disadvantages of their use in clinical settings are the limited number of HSCs present within each UCB unit1. The insufficient number of HSCs results in delayed engraftment and hematopoietic recovery, risk of graft rejection, and aberrant immune reconstitution.
Currently, various methods and strategies have been developed to ex vivo expand the limited number of HSCs from UCBs. Combinations of different cytokine cocktails with small molecules or compounds in ex vivo cultures result in various degrees of expansion in HSC numbers2,3,4,5,6,7,8. Importantly, ex vivo culture conditions induce stress, leading to rapid cell proliferation, increased metabolic activity and loss of the primitive characteristics that define primary HSCs. Therefore, developing protocols that lead to expansion of a great number of functional HSCs with characteristics that closely resemble primary primitive HSCs are needed.
Serum-free cultures of CD34+ cells isolated from UCBs and treated with valproic acid (VPA) result in the expansion of large numbers of primitive HSCs4,9,10. The HSC expansion is not solely due to the proliferation of the existing HSCs derived from UCBs. Instead, this expansion is due to the acquisition of a primitive phenotype combined with a limited number of cell divisions and proliferation9. Within the initial 24&#8211;48 h of incubation with a combination of cytokines and VPA, CD34+ cells acquire a transcriptomic and phenotypic profile that characterize long-term HSCs. The significant increase in the percentage of HSCs is accompanied by a prompt increase in the number of HSCs (63 fold increase within 24 h of VPA treatment)9. Notably, the VPA-ex vivo expansion strategy expands HSCs with low metabolic activity, which further highlights their primitive characteristics.
The method described here provides conditions and treatments that lead to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications. This ex vivo expansion strategy uses a cytokine cocktail combined with VPA treatment. VPA is an FDA approved drug for treatment of bipolar disorders and other neurological diseases. The HSC expansion with VPA is prompt and occurs within 7 days, minimizing both the time of manipulation and the risk of contamination. Importantly, this protocol allows for the acquisition of long-term HSC phenotypic markers such as CD90 and CD49f within 24-48 h following treatment with VPA9. Expanded HSC grafts created with this protocol have the capacity to regenerate the hematopoietic system since they can differentiate into all hematopoietic cell lineages and establish long-term engraftment following transplantation into myeloablated NSG mice&#160;models4. Moreover, this protocol is highly reproducible and allows for efficient and rapid isolation of viable CD34+ cells from UCBs, which is critical for industrialization of this procedure.
The VPA ex vivo expansion protocol also has the potential to overcome the significant loss of HSCs which occurs during gene editing11. Gene editing requires exposure to cytokines, which are necessary for cycling cells and activation of DNA-repair mechanisms. Due to the prompt effects of VPA treatment, this method might be beneficial for the generation of a higher number of genetically modified cells within a period of time that is relevant for currently utilized gene modification protocols.

<table>
	<tbody>
		<tr>
			<td>Expansion Media</td>
			<td>Sigma-Aldrich</td>
			<td>Stemline II stem cell expansion media S0192-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>AO/PI (acridine orange / propidium iodide) staining solution for live/dead Mammalian nucleated cells.</td>
			<td>Nexcelom</td>
			<td>CS2-0106-25mL</td>
			<td></td>
		</tr>
		<tr>
			<td>APC Mouse Anti-Human CD34 Clone 581</td>
			<td>BD BIOSCIENCE</td>
			<td>555824</td>
			<td></td>
		</tr>
		<tr>
			<td>APC Mouse IgG1, &#954; Isotype Control Clone MOPC-21</td>
			<td>BD BIOSCIENCE</td>
			<td>555751</td>
			<td></td>
		</tr>
		<tr>
			<td>autoMACS Rinsing Solution</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-091-222</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Falcon 15 ml Tube</td>
			<td>BD Biosciences</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Falcon 5 ml Polystyrene Round-Bottom Tube</td>
			<td>BD Biosciences</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Falcon 50 ml Tube</td>
			<td>BD Biosciences</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumine solution</td>
			<td>Sigma-Aldrich</td>
			<td>A8412</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 MicroBead Kit, contain cd34 beads and fcr blocking reagent, human</td>
			<td>Miltenyi Biotec</td>
			<td>130-046-703</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell analyzer</td>
			<td>BD Biosciences</td>
			<td>FACS Canto II</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell analyzer and sorter</td>
			<td>BD Biosciences</td>
			<td>FACS Aria II</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>Nexcelom</td>
			<td>Cellometer Auto 2000 Cell Viability Counter</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell separator device</td>
			<td>autoMACS Pro Separator Miltenyi Biotec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Counting Chambers</td>
			<td>Nexcelom</td>
			<td>CHT4-PD100-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Density gradient media</td>
			<td>GE Healthcare Bio-Sciences AB</td>
			<td>17-1440-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene diamine tetra-acetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E8008-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-human CD90 (Thy1) Clone 5E10</td>
			<td>BIOLEGEND</td>
			<td>328108</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Mouse IgG1, &#954; Isotype Ctrl (FC) Antibody</td>
			<td>BIOLEGEND</td>
			<td>400109</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Corning cellgro</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Flt-3 Ligand Protein</td>
			<td>R&#38;D Systems</td>
			<td>308FKN</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IL-3 Protein (IL-3)</td>
			<td>R&#38;D Systems</td>
			<td>203-IL</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human SCF Protein</td>
			<td>R&#38;D Systems</td>
			<td>255-SC</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Thrombopoietin Protein (TPO)</td>
			<td>R&#38;D Systems</td>
			<td>288-TP</td>
			<td></td>
		</tr>
		<tr>
			<td>Total Antibody Compensation Bead Kit</td>
			<td>thermofisher</td>
			<td>A10497</td>
			<td></td>
		</tr>
		<tr>
			<td>Umbilical Cord-blood</td>
			<td>Placental Blood Program at the New York Blood Center</td>
			<td>http://nybloodcenter.org/products-services/blood-products/national-cord-blood-program/cord-blood-101/</td>
			<td></td>
		</tr>
		<tr>
			<td>Valproic acid sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>P4543</td>
			<td></td>
		</tr>
	</tbody>
</table>

ex vivo expansion of hematopoietic stem cells from human umbilical cord blood-derived cd34+ cells using valproic acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

The ex vivo protocol described here increases the number of primitive HSCs generated from CD34+ cells isolated from UCBs (Figure 1). Priming of CD34+ cells for 16 h with a cytokine cocktail, followed by treatment with VPA for an additional 7 days, leads to a great degree of HSC expansion. Remarkably, the pool of expanded cells is highly enriched for HSCs, which are phenotypically defined by CD34+CD90+ markers. The t...

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Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

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9.7K Views.  Icahn School of Medicine at Mount Sinai. Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

Video: Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual capabilities to the degenerated retina. Nevertheless, progress in cell replacement therapy presently faces the challenges of requiring an off-the-shelf source of high quality and standardized human retinas. Therefore, an easy and stable protocol is needed for the experiments. Here, we develop an optimized protocol, based on a self-organizing method with the use of exogenous molecules and reagent A as well as manual excision to generate the three-dimensional human retina organoids (RO). The human Pluripotent Stem Cells (PSCs)-derived RO expresses specific markers for photoreceptors. With the addition of COCO, a multifunctional antagonist, the differentiation efficiency of photoreceptor precursors and cones is significantly increased. The efficient use of this system, which has the benefits of cell lines and primary cells, and without the sourcing issues associated with the latter, could produce confluent retinal cells, especially photoreceptors. Thus, the differentiation of PSCs to RO provides an optimal and biorelevant platform for disease modelling, drug screening and cell transplantation.

Using a self-organizing method, we develop a protocol with the addition of COCO that could significantly increase the generation of photoreceptors.

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual ...

Isolation and Magnetic Separation of Murine CD34+ Stem Cells from the Vascular Wall

Immunomagnetic Isolation of the Vascular Wall-Resident CD34+ Stem Cells from Mice

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury and repair. Establishing a stable and efficient method to culture functional murine CD34+ VW-SCs is essential for further investigating the mechanisms involved in the proliferation, migration, and differentiation of these cells under various physiological and pathological conditions. The described method combines magnetic bead screening and flow cytometry to purify primary cultured resident CD34+ VW-SCs. The purified cells are then functionally identified through immunofluorescence staining and Ca2+ imaging. Briefly, vascular cells from the adventitia of the murine aorta and mesenteric artery are obtained through tissue block attachment, followed by subculturing until reaching a cell count of at least 1 &#215; 107. Subsequently, CD34+ VW-SCs are purified using magnetic bead sorting and flow cytometry. Identification of CD34+ VW-SCs involves cellular immunofluorescence staining, while functional multipotency is determined by exposing cells to a specific culture medium for oriented differentiation. Moreover, functional internal Ca2+ release and external Ca2+ entry is assessed using a commercially available imaging workstation in Fura-2/AM-loaded cells exposed to ATP, caffeine, or thapsigargin (TG). This method offers a stable and efficient technique for isolating, culturing, and identifying vascular wall-resident CD34+ stem cells, providing an opportunity for in vitro studies on the regulatory mechanisms of VW-SCs and the screening of targeted drugs.

Author Spotlight: Enhanced Isolation and Characterization of Vascular Wall Resident Murine CD34+ Stem Cells Using Magnetic Bead Screening-Coupled Flow Cytometry

This study has established a stable and efficient method for the isolation, culture, and functional determination of vascular wall-resident CD34+ stem cells (CD34+ VW-SCs). This easy-to-follow and time-effective isolation method can be utilized by other investigators to study the potential mechanisms involved in cardiovascular diseases.

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury ...

Dissection and Isolation of WJ-MSCs from the Human Umbilical Cord

Isolation of Umbilical Cord-Derived Mesenchymal Stem Cells with High Yields and Low Damage

Mesenchymal stem cells (MSCs) are a population of multipotent cells with remarkable regenerative and immunomodulatory properties. Wharton's jelly (WJ) from the umbilical cord (UC) has gained increasing interest in the biomedical field as an outstanding source of MSCs. However, challenges such as limited supply and lack of standardization in existing methods have arisen. This article presents a novel method for enhancing MSC yield by dissecting intact WJ from the umbilical cord. The method employs blunt dissection to remove the epithelial layer, maintaining the integrity of the entire WJ and resulting in an increased quantity and viability of harvested MSCs. This approach significantly reduces WJ waste compared to conventional sharp dissection methods. To ensure the purity of WJ-MSCs and minimize external cellular influence, a procedure utilizing internal tension to peel off the endothelium after flipping the UC was conducted. Additionally, the Petri dish was inverted for a short time during explant culture to improve attachment and cell outgrowth. Comparative analysis demonstrated the superiority of the proposed method, showing a higher yield of WJ and WJ-MSCs with better viability than traditional methods. The similar morphology and expression pattern of cell surface markers in both methods confirm their characterization and purity for various applications. This method provides a high-yield and high-viability approach for WJ-MSC isolation, demonstrating great potential for the clinical application of MSCs.

Author Spotlight: Advancements in Stem Cell-Derived Vascularized Organoids and Optimized Isolation of Wharton's Jelly for Enhanced MSC Production

This study presents a unique blunt dissection procedure to preserve the integrity of Wharton's jelly (WJ), resulting in less damaged WJ and a greater quantity and viability of the harvested mesenchymal stem cells (MSCs). The method demonstrates superior yield and proliferative ability compared to conventional sharp dissection methods.

Mesenchymal stem cells (MSCs) are a population of multipotent cells with remarkable regenerative and immunomodulatory properties. Wharton's jelly (WJ) ...