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

Summary

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.

Abstract

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.

Introduction

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 a....

Protocol

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

1. 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 °C.
2. Warm media at room tempera.......

Discussion

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.......

Materials

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