Daniel R. Marshak

Post-translational Modifications in Cartilage Oligomeric Matrix Protein CHARACTERIZATION OF THE N-LINKED OLIGOSACCHARIDES BY MATRIX-ASSISTED LASER DESORPTION IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY

Analysis of the carboxymethylated subunit of human cartilage oligomeric matrix protein (COMP) by matrix-assisted laser desorption time-of-flight mass spectrometry indicated a protonated molecular mass of 86949 ± 149 Da, compared with 83547.0 Da calculated from the sequence. Treatment withN-glycanase caused a reduction in mass of 3571 ± 219 Da, but there was no loss of mass after treatment withO-glycanase or neuraminidase. Peptides containing two putative sites of N-glycosylation were purified and characterized. Analysis of the masses of these afterN-glycanase treatment indicated that one was substituted at Asn-101 with an oligosaccharide of mass 1847.2 ± 6.6 Da, and the other was unsubstituted at Asn-124. The remaining site of attachment, at Asn-721, was, therefore, also substituted with an oligosaccharide of mass 1724 ± 226 Da. Analysis of the total monosaccharide content by chemical methods indicated that there were no additional oligosaccharide substituents. The MALDI-TOF mass spectra of COMP from bovine fetal and adult cartilage were compared, indicating a more heterogeneous pattern of substitution at Asn-101 in the fetal form. Since COMP is distributed throughout the pericellular and territorial environments in developing cartilage but occupies the interterritorial zone in mature cartilage, these changes in glycosylation may allow for different intermolecular interactions.

16 Mesenchymal Stem Cells of Human Adult Bone Marrow

We define stem cells broadly as those cells that give rise to progeny with more than one differentiated phenotype and that may be greatly expanded in an undifferentiated form. This differs from a “progenitor cell,” which gives rise to a single cell lineage only. Human mesenchymal stem cells (hMSCs) are isolated from bone marrow and expanded ex vivo. Flow cytometry using many different surface markers has demonstrated the expanded population to be >98% homogeneous and in defined in vitro assays these cells readily differentiate to multiple connective tissue lineages, including osteoblasts, chondrocytes, and adipocytes (Fig. 1) (Pittenger et al. 1999). In vivo implantation of these cells at orthotopic sites will also yield tissues in these lineages. Additionally, cultured hMSCs either produce, or can be induced to produce, cytokines for support of hematopoietic cells (Majumdar et al. 1998; Cheng et al. 2000). Cocultures of the MSCs with hematopoietic stem cells (HSCs) demonstrated that hMSCs or adipogenic hMSCs can support the in vitro maintenance, and even expansion, of HSCs, suggesting hMSCs serve as functional stroma (Thiede et al. 2000). In addition, conditions that produce myogenic differentiation of rat MSCs have been reported (Saito et al. 1995), and hMSCs show similar behavior but perhaps less efficiently. MSCs with similar potential have been isolated from other species as well, and those isolated from rabbits differentiated to tenocytes to produce a suitable replacement for severed tendon with excellent biomechanical stability (Young et al. 1998). The rabbit MSCs were also shown to form suitable cartilaginous grafts...

Preface/Front Matter

The field of stem cell research has attracted many investigators in the past several years. Progress in embryology, hematology, neurobiology, and skeletal biology, among many other disciplines, has centered on the isolation and characterization of stem cells. The approaching completion of the sequencing of the human genome has lent further impetus to exploring how gene expression in stem cells relates to their dual functions of self-renewal and differentiation. Two small meetings held at the Banbury Center of Cold Spring Harbor Laboratory in 1996 and 1999 served to bring together groups of scientists eager to discuss the role of stem cells in development, tissue homeostasis, and regeneration. These meetings highlighted both the quickening pace of discovery relating to the basic biology of stem cells and the increasing scope for their clinical exploitation. They also convinced us that it was timely to assemble a monograph that would help to make the fundamentals of stem cell biology more accessible to those seeking better acquaintance with the subject. We thank Inez Sialiano, Pat Barker, Danny deBruin, and John Inglis of the Cold Spring Harbor Laboratory Press for enabling this project to be realized. We also acknowledge the efforts of the entire staff of the Press who contributed to the editing and production process. Drs. James Watson, Bruce Stillman, and Jan Witkowski were highly supportive of this enterprise. A particular note of thanks is due Mr. James S. Burns for his encouragement and enthusiasm, as well as his vision and accomplishments, in both the development

1 Introduction: Stem Cell Biology

STEM CELLS: AN OVERVIEW There is still no universally acceptable definition of the term stem cell, despite a growing common understanding of the circumstances in which it should be used. According to this more recent perspective, the concept of “stem cell” is indissolubly linked with growth via the multiplication rather than the enlargement of cells. Various schemes for classifying tissues according to their mode of growth have been proposed, one of the earliest of which is that of Bizzozero (1894). This classification, which relates to the situation in the adult rather than in the embryo, recognizes three basic types of tissues: renewing, expanding, and static. Obvious examples of the first are intestinal epithelium and skin, and of the second, liver. The third category was held to include the central nervous system, although recent studies have shown that neurogenesis does continue in adulthood, for example, with regard to production of neurons that migrate to the olfactory bulbs (Gage 2000). There are various problems with such schemes of classification including, for instance, assignment of organs like the mammary gland which, depending on the circumstances of the individual, may engage in one or more cycles of marked growth, differentiation, and subsequent involution. Any attempt to find a universally acceptable definition of the term stem cell is probably doomed to fail. Nonetheless, certain attributes can be assigned to particular cells in both developing and adult multicellular organisms that serve to distinguish them from the remaining cells of the tissues to which they belong. Most...