Ex vivo expansion of cord blood mononuclear cells on mesenchymal stem cells

doi:10.1080/14653240410004871

Cytotherapy

Volume 6, Issue 4, August 2004, Pages 311-317

https://doi.org/10.1080/14653240410004871 Get rights and content

Background

Cord blood (CB) cells are being used increasingly as a source of hematopoietic cells to support high dose chemotherapy. However, CB units contain low numbers of cells, including CD34 cells, and thus their use is associated with significant delays in engraftment of neutrophils and platelets. Ex vivo expansion of CB has been proposed to increase the numbers of cells available. We and others have reported the requirement of CD34 selection for optimal expansion of CB products'; however, the selection of frozen CB products in clinical trials results in significant loss of CD34⁺ cells, with a median recovery of 50, but less than 40% recovery in more than one-third of products. In the present studies we evaluated the potential of mesenchymal stem cells (MSC) to support ex vivo expansion of unselected CB products.

Methods

Mononuclear cells (MNC) from CB products were isolated and cultured on preformed MSC layers in T1 50 flasks containing 50 mL Stemline II media plus hematopoietic growth factors. Various culture conditions were compared for optimal expansion of the CB MNC.

Results

Ex vivo expansion of CB MNC on MSC resulted in 10- to 20-fold expansion of total nucleated cells, seven- to 18-fold expansion of committed progenitor cells, two- to five-fold expansion of primitive progenitor cells and 16- to 37-fold expansion of CD34 cells.

Discussion

These studies demonstrated significant expansion of CB products without CD34 cell selection using culture conditions that are clinically applicable. Our current focus is to initiate clinical trials to evaluate the in vivo potential of CB cells expanded with these conditions.

Introduction

Human umbilical cord blood (CB) represents a unique source of transplantable hematopoietic cells. The number of patients transplanted with CB, to provide hematopoietic reconstitution following high dose chemotherapy (HDC), has rapidly increased over the past several years. The use of CB products for unrelated allogeneic recipients offers a number of advantages over the use of BM from unrelated donors, including rapid access to products, and thus a decreased delay in the time period to transplant; increased representation of minority groups in CB banks compared with BM registries; and the potential for decreased GvHD and the use of CB grafts with more HLA disparity than is acceptable with BM allografts. However, the numbers of progenitor cells in CB allografts are lower than obtained for PBPC products or BM grafts. Therefore several groups [1,2], including our own [3], have evaluated ex vivo expansion of CB products in an attempt to increase the number of progenitor cells available for transplant. These expanded products have been used successfully to engraft cancer patients after HDC. The current clinical conditions for ex vivo expansion of CB cells require selection of the CD34⁺ subset or culture in perfusion systems, as unfractionated or even mononuclear cells (MNC) do not expand well in static culture [4]. CB products destined for clinical use are frozen. CD34⁺ cell selection of the thawed units often results in low CD34⁺ cell recoveries and suboptimal purities, resulting in lower expansion overall. It would be advantageous to develop culture conditions that would enable ex vivo expansion of CB MNC and provide increased numbers of cells for transplantation.

A number of previous studies have demonstrated the supportive role of stromal cells for hematopoietic stem cells and progenitor cells [5,6]. In addition, recent clinical studies have evaluated the potential of mesenchymal stem cells (MSC) to facilitate engraftment and possibly decrease the incidence of GvHD. We therefore evaluated the ability of MSC to support the expansion of CB MNC and propose clinical studies that would incorporate the expanded cells plus MSC. The data presented in this paper demonstrate that MNC from CB products can be expanded efficiently when cultured on MSC, resulting in a ten fold increase in the number of cells for transplantation.

Section snippets

Ex vivo expansion of CD34 selected CB products

The details of the clinical studies of ex vivo expanded CB products have been reported in detail previously [3]. All CB products were thawed using the procedure developed by Rubinstein et al [7]. The fraction to be ex vivo expanded was CD34-selected using the Isolex 300i device (Baxter Healthcare, Deerfield, IL), following the manufacturers' recommended procedures. Following selection the CD34⁺product was evaluated for the number of CD34⁺ cells by flow cytometric analysis and the product was

CD34 selection of CB products

Previous studies have demonstrated that ex vivo expansion of CB products is optimal starting with CD34-selected products [4]. For clinical studies the use of clinical selection devices is mandated by the FDA and therefore we used the Isolex 300i for studies evaluating the potential of ex vivo expanded CB products in cancer patients [3]. The results of CD34 selection for 30 patients are presented in Table 1. For these studies 40% or 60% fractions of the CB products were selected and expanded.

Acknowledgements

The authors would like to thank Dr Pablo Rubinstein and Cladd Stevens from the New York Cord Blood Bank for supply of cord blood products. In addition, we thank Frank Swartwelder, Bruce Lehr and Laurie Donahue from Sigma-Aldrich for supply of Stemline II expansion media.

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Ex vivo expansion of cord blood mononuclear cells on mesenchymal stem cells

Background

Methods

Results

Discussion

Introduction

Section snippets

Ex vivo expansion of CD34 selected CB products

CD34 selection of CB products

Acknowledgements

BBMT

Blood

BBMT

Blood

A phase I trial of augmentation of unrelated umbilical cord blood transplantation with ex-vivo expanded cells

Blood

Umbilical cord blood transplants in adults using a combination of unexpanded and ex vivo expanded cells: preliminary clinical observations

Blood