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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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.......

Representative Results

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.......

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.......

Acknowledgements

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

....

Materials

NameCompanyCatalog NumberComments
Expansion MediaSigma-AldrichStemline II stem cell expansion media S0192-500ML
AO/PI (acridine orange / propidium iodide) staining solution for live/dead Mammalian nucleated cells.NexcelomCS2-0106-25mL
APC Mouse Anti-Human CD34 Clone 581BD BIOSCIENCE555824
APC Mouse IgG1, κ Isotype Control Clone MOPC-21BD BIOSCIENCE555751
autoMACS Rinsing SolutionMACS Miltenyi Biotec130-091-222
BD Falcon 15 ml TubeBD Biosciences352097
BD Falcon 5 ml Polystyrene Round-Bottom TubeBD Biosciences352063
BD Falcon 50 ml TubeBD Biosciences352098
Bovine serum albumine solutionSigma-AldrichA8412
CD34 MicroBead Kit, contain cd34 beads and fcr blocking reagent, humanMiltenyi Biotec130-046-703
Cell analyzerBD BiosciencesFACS Canto II
Cell analyzer and sorterBD BiosciencesFACS Aria II
Cell counterNexcelomCellometer Auto 2000 Cell Viability Counter
Cell separator deviceautoMACS Pro Separator Miltenyi Biotec
Counting ChambersNexcelomCHT4-PD100-002
Density gradient mediaGE Healthcare Bio-Sciences AB17-1440-03
Ethylene diamine tetra-acetic acid (EDTA)Sigma-AldrichE8008-100ML
FITC anti-human CD90 (Thy1) Clone 5E10BIOLEGEND328108
FITC Mouse IgG1, κ Isotype Ctrl (FC) AntibodyBIOLEGEND400109
Inverted microscope
PBSCorning cellgro21-040-CV
Penicillin-StreptomycinThermo Fisher Scientific15140122
Recombinant Human Flt-3 Ligand ProteinR&D Systems308FKN
Recombinant Human IL-3 Protein (IL-3)R&D Systems203-IL
Recombinant Human SCF ProteinR&D Systems255-SC
Recombinant Human Thrombopoietin Protein (TPO)R&D Systems288-TP
Total Antibody Compensation Bead KitthermofisherA10497
Umbilical Cord-bloodPlacental Blood Program at the New York Blood Centerhttp://nybloodcenter.org/products-services/blood-products/national-cord-blood-program/cord-blood-101/
Valproic acid sodium saltSigma-AldrichP4543

References

  1. Broxmeyer, H. E. Enhancing the efficacy of engraftment of cord blood for hematopoietic cell transplantation. Transfusion and Apheresis Science. 54 (3), 364-372 (2016).
  2. Boitano, A. E., et al.

Explore More Articles

Ex Vivo ExpansionHematopoietic Stem CellsUmbilical Cord BloodCD34 CellsValproic AcidRegenerative MedicineTransplantation TherapyCytokine CocktailGene EditingCell SeparationMononuclear CellsDensity GradientPurification

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