HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by van Bekkum, D. W. Search for Related Content PubMed PubMed Citation Articles by van Bekkum, D. W.

New Stem Cell Workshop

Leiden University, Molecular Cell Biology, Leiden, The Netherlands

Dirk W. van Bekkum, M.D., Ph.D., Crucell B.V., P.O. Box 2048, 2301 CA Leiden, The Netherlands. Telephone: 31-71-5248787; Fax: 31-71-5248846; e-mail: bekkum{at}crucell.com

On September 18 and 19, 2000, more than 120 scientists from all over the world convened in Leiden, the Netherlands, to participate in a workshop on the plasticity of stem cells from adult tissues. The occasion was in celebration of the completion of the new building of the Dutch biotech company, Crucell. This meeting included wet workshops, i.e., demonstrations in the laboratory of the various quite sensational recent discoveries. The opportunity to organize such demonstrations arose from the availability of equipped but not occupied laboratories just prior to the move from the old premises. The meeting was funded jointly by Crucell and the Comprehensive Cancer Center at Rotterdam.

P. Quesenberry (Boston) described changes in engraftment properties with various stages of the cell cycle of mouse hematopoietic stem cells. Bone marrow from male mice was cultured in the presence of growth factors and used at various intervals for competitive engraftment experiments in irradiated female recipients. The cycle parameters were determined for lineage-rhodamine^low Hoechst^low stem cells that were purified and then cultured separately under identical conditions. Competitive grafting capacity of the bone marrow as determined by Southern blot analysis revealed a decrease during late S and M phases and an increase again during G₁ and early S phases. These findings are highly suggestive of reversible changes in the phenotype of stem cells as they move through the cell cycle, but some uncertainty remains because the two properties that were compared had to be determined in separate cell populations.

S. Knaan-Shanzer (Leiden) provided evidence for a reversal of differentiation of primitive hematopoietic stem cells as defined by current surface markers using human cord-blood-derived cells. Within the CD34 population, the early lineage markers' negative (ELM^–) subpopulation, defined by these authors as CD34⁺⁺, CD33, CD38, CD71^–, can repopulate the bone marrow of non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, while the ELM⁺ population cannot, and was therefore considered to be more differentiated. Following culturing on stroma in the absence of serum, the latter cells generated ELM^– cells and acquired repopulating ability. The authors consider that this finding challenges the concept of hierarchy within the primitive stem cell compartment and favors the idea of stem cell plasticity.

Under different conditions (fetal bovine serum and growth factors), C. Dorrell and F. Mazurier from John Dick's laboratory in Toronto demonstrated a large expansion of CD34⁺38^– cells in cord blood cultures without an increase in NOD/SCID repopulating capacity. These observations suggest a dissociation between surface markers and functional phenotype but would also fit the hypothesis that repopulating stem cells constitute an as yet unidentified subpopulation of lineage^– cells.

The session on neural stem cells focused on the isolation of these cells from adult mouse brain. These cells are most prevalent in the walls of the lateral ventricles and may form colonies in vitro, the so-called neurospheres. After a certain period of culture, the neural cells derived from Rosa mice (these mice are transgenic for LacZ, which encodes for the marker E. coli beta-galactosidase) were grafted into chicken or mouse embryos by J. Frisèn et al. (Copenhagen). They demonstrated their grafting technique into the amniotic cavity of stage 3 chick embryos and reported that adult neural stem cells can integrate into both chicken and mouse embryos to contribute to the generation of tissues and organs of all germ layers. The development was not pursued beyond the embryonic stages.

D. Herrera (formerly with A. Vescovi in Milano and presently at Columbia University, New York), demonstrated the dissection procedure for obtaining neuronal stem cells from the subventricular region of the forebrain of mice. These cells, when injected into sublethally irradiated recipients, were shown to repopulate the blood-forming tissues to an average of 40% of donor-derived cells of various lineages.

Using his well-known fetal sheep model, E. Zanjani (Salt Lake City) demonstrated the presence of human hepatocytes in the livers of sheep that were grafted with human neuronal stem cells in utero. He also reported the occurrence of human hepatocytes after transplanting human cord blood or bone marrow cells.

Along with these findings were the demonstrations of hepatocytes with donor characteristics in the liver of human bone marrow transplant recipients. M. Allison and R. Poulson (London) identified male hepatocytes in the liver of female recipients of a male bone marrow transplant, as well as in the female liver transplant in male recipients. The percentage of hepatocytes of "bone marrow origin" was low, but they sometimes occurred in small clusters, suggesting a clonal origin.

N. Theise (New York) and D. Krause (New Haven) performed an elaborate quantitative assessment of the number of Y chromosome positive hepatocytes as well as cholangiocytes in liver sections of similar human liver or bone marrow transplant recipients. Approximately 4%-40% of both cell types were scored as Y positive and should therefore have been derived from hematopoietic stem cells, the highest values being found in a patient who suffered from recurrent hepatitis. They also studied the livers of mouse hematopoietic chimeras at various times after transplantation of bone marrow or purified stem cells and recorded between 1% and 2% of the hepatocytes to be donor-derived.

B. Petersen (Gainesville) applied hepatic injury in established male-to-female rat hematopoietic chimeras. At day 13 after the injury, 0.14% of the hepatocytes were found to be Y positive. He also identified recipient-derived oval cells in transplanted rat livers. These can only be derived from nonhepatic tissue. It is of interest that oval cells of the liver were found to carry the hematopoietic stem cell marker Thy-1. Even more striking evidence of the ability of hematopoietic stem cells to generate hepatocytes was provided by M. Grompe (Portland), who uses fumarylacetoacetate hydrolase (FAH)-deficient mice. These animals suffer from tyrosinemia type I and can only survive progressive liver failure if treated with 2—(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexanedione (NTBC). These mice were lethally irradiated and grafted with wild-type Rosa (LacZ-positive) bone marrow. Three weeks after transplantation, the NTBC treatment was discontinued, and approximately 50% of the mice survived until analyzed 6 months later. The livers contained 30%-50% FAH-positive LacZ-positive hepatocytes. These were located in multiple nodules, suggesting multiclonal origin. Liver function was largely normal but reconstitution was not as complete as following transplantation of wild-type hepatocytes.

Several investigators presented evidence that cells from the bone marrow can give rise to muscle cells. Y. Matzusaki and E. Kotsopoulou from the laboratory of R. Mulligan (Boston) demonstrated the characterization by fluorescence-activated cell sorting (FACS) methodology of the subpopulation of bone marrow cells that populate the skeletal muscles of lethally irradiated mdx mice with dystrophin positive myofibers.

Using the model of cardiotoxin-induced muscle damage in mice, F. Mavilio (Milano) demonstrated the occurrence of bone-marrow-derived nuclei in a small proportion of the regenerating myofibers. These muscle precursors were shown to be derived from adherent as well as nonadherent bone marrow fractions in experiments where the cells were injected directly into the damaged muscle area.

R. Bittner and F. Wagner (Vienna) showed the presence of Y chromosomes not only in nuclei of the skeletal muscles but also in nuclei of cardiac muscle cells of dystrophic (mdx) female mouse recipients of normal male donor mice. In contrast, this phenomenon was not observed in the muscles of normal female recipients.

C. Verfaillie (Minnesota) showed how to isolate multipotent adult progenitor cells from human bone marrow and how to expand and manipulate these cells by culturing them in the presence of various growth factors to differentiate into mesenchymal, visceral, mesodermal, neuroectodermal, and endodermal cell types. Cell type characterizations were established with immunohistochemistry and Western blotting of proteins specifically expressed in each type of cell.

The isolation of one of these precursor cells from human (non-stromal) bone marrow was reported by M. Long (Ann Arbor). These osteoprogenitor cells require specific bone regulatory cytokines such as bone morphogenic protein for expansion and differentiation. In contrast to mouse bone marrow, these progenitors are not present in human bone marrow stroma.

During the morning sessions of the two-day meeting, faculty presented a brief outline of their findings and of the techniques employed. These presentations served as introduction to the demonstrations that were given during each whole afternoon. These included microscopical inspection of histological slides, infusion, dissection, isolation, cell harvesting techniques, and FACS analysis and sorting. The key papers of the contributors had been distributed among all participants some time before the meeting, and elaborate protocols describing the many new techniques were distributed during the meeting. All this paved the way for very open detailed discussions and allowed serious scrutiny of these surprising new developments.

The general conclusion, as admirably formulated by I. Lemischka (Princeton) in his summing up of the meeting, was that something wonderful and unprecedented is indeed going on. Perhaps the title of the workshop was somewhat misleading, as it is actually new potentials of the old stem cells that are being discovered, thanks to new methods of culture and detection not available before. Although pluripotentiality of adult stem cells across several tissue specificities seems to be established, it is yet far from certain if full functionality comes along with phenotypic transdifferentiation. Also, proof of the clonal origin of these stem cells is still needed in many cases. However, the excitement that these discoveries have evoked seems justified: a whole new era of stem cell research has begun, which will involve many more tissues of the mammalian organism than the hematoimmunological system.

The stakes for medical applications are high: are adult stem cells to compete with or even to replace the much-debated embryonic and fetal stem cells? The promise that every person may carry his or her own compatible donor cells justifies large investments in this new and exciting challenge.

Received February 21, 2000; accepted for publication March 15, 2001.

This article has been cited by other articles:

				N. Forraz, R. Pettengell, and C. P. McGuckin Characterization of a Lineage-Negative Stem-Progenitor Cell Population Optimized for Ex Vivo Expansion and Enriched for LTC-IC Stem Cells, January 1, 2004; 22(1): 100 - 108. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by van Bekkum, D. W. Search for Related Content PubMed PubMed Citation Articles by van Bekkum, D. W.