In January 1990, at the beginning of the Human Bone Marrow Stem Cell Concerted Action (CA), no laboratory in Europe was planning to develop large scale purification methods for isolating the hematopoietic stem cell for either cell or gene therapy.
In 1993, staff in various European laboratories have become acquainted, and have interacted with each other. This has allowed us to organize stem cells, to meet regularly and to compare various clinical devices to purify stem cells, and to organize gene therapy or clinical groups at a European level.
Various European laboratories have interacted to organize stem cell banks and to compare various clinical devices to purify stem cells for use in gene therapy. Media have been developed in for use in multicentre clinical trials in order to standardize stem cell assays which are a prerequisite for stem cell purification.
The mixed colony assay is an assay where one cell is able to provide a colony in which the megakaryocytes, the granulocytes, the monocytes and erythrocytes are able to develop simultaneously in a clone. Some groups are developing a modification of this assay for the production of T or B lymphoid lineage cells together with the myeloid lineages. Standardized media for mixed colony assays have been provided with all the recombinant cytokines available to date. One laboratory has developed this assay in serum free conditions. With this medium very large colonies may be obtained from a single cell. Liquid cultures have also been developed in order to expand earlyprogenitors for the mass production of cells for transfusion or for favoring rapid engraftment after transplantation. An animal model for a human stem cell assay is also being developed using immunodeficient mice engrafted with hemopoietic tissues and treated with human cytokines.
Various laboratories have used bone marrow cells to purify CD34+ stem cells but large amounts of bone marrow are not always available. Umbilical cord blood was found to be another source of cells. The stem cells obtained here have a larger haematopoietic potential than those obtained in adult bone marrow or adult peripheral blood, and although the volume of blood in the umbilical cord is smaller than the volume of marrow which is used for adult bone marrow transplantation, there are sufficient umbilical cord blood cells for adult transplantation. This has been demonstrated in vitro and is being used in hospitals for transplantation.
Various groups are developing new vectors and new methods for the stable introd uction of genes of interest into the genome in various haematological diseases. The possibility of gene therapy using stem cells would require only one transplant and one difficulty that has been overcome is the problem of inserting genes into cells which are not dividing. Stem cells are in the Go phase or in a quiescent state and by releasing the Go block the stable transfer of marker genes has been significantly improved.
In 1990, when the CA started, human bone marrow stem cells were not being purified for clinical use. These cells are very rare, representing less than one in 10000 mononuclear cells in the human bone marrow. These cells are very difficult to purify because they are not easy to assay. No simple assay is available for detecting single stem cells. The best assay has been developed in the murine system. In this system, 20 purified stem cells, when injected into an irradiated mouse, were able to reconstitute hematopoiesis completely. Such experiments are impossible in man. This is why the CA has developed a quick in vitro assay to identify the 'stem cell compartment'. With their improved identification (the CD34+, CD38-, DR-, Thy-1+ cells), we have compared and improved the various purification methods which are available for laboratory work. We have chosen among these methods, those which could be used easily for clinical studies. We have compared not only the efficacy of various devices but also their cost and the fact that they are easy to use. During the CA, umbilical cord blood cells have been identified as a new and rich source of stem cells. As bone marrow donors are too few for the needs of transplantation, it was important to develop this new approach. Umbilical cord blood provides an unlimited source of cells which covers all HLA types. Moreover umbilical cord blood is easier to collect than bone marrow, and is unlimited.
Stem cell transplantation has a very wide range of uses for future studies. It will allow cancer treatments to be carried out with higher doses of chemotherapy or radiotherapy, with the new stem cells being grafted after the treatment. It will also be used for the introduction of new genes for gene therapy. The stem cell is indeed an excellent candidate for gene therapy as this cell generates the cells of the hematopoietic and immune system.