Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells

Abstract

Marrow stromal cells are adult stem cells from bone marrow that can differentiate into multiple nonhematopoietic cell lineages. Previous reports demonstrated that single-cell-derived colonies of marrow stromal cells contained two morphologically distinct cell types: spindle-shaped cells and large flat cells. Here we found that early colonies also contain a third kind of cell: very small round cells that rapidly self-renew. Samples enriched for the small cells had a greater potential for multipotential differentiation than samples enriched for the large cells. Also, the small cells expressed a series of surface epitopes and other proteins that potentially can be used to distinguish the small cells from the large cells. The results suggested it will be important to distinguish the major subpopulations of marrow stromal cells in defining their biology and their potential for cell and gene therapy.B one marrow contains at least two kinds of stem cells, hematopoietic stem cells and stem cells for nonhematopoietic tissues (1-26), variously referred to as mesenchymal stem cells or marrow stromal cells (MSCs). MSCs are of interest because they are easily isolated from a small aspirate of bone marrow and readily generate single-cell-derived colonies (1-5, 25, 27); the single-cell-derived colonies can be expanded through as many as 50 population doublings in about 10 weeks (25), and they can differentiate into osteoblasts, adipocytes, chondrocytes (1-13), myocytes (9), astrocytes, oligodendrocytes, and neurons (17,23,26,27). For these reasons, the cells are currently being tested for their potential use in cell and gene therapy for a number of human diseases (22,24). Previous reports (3,11,12) demonstrated that single-cell-derived colonies of human MSCs are heterogeneous in that they contain at least two morphologically distinct kinds of cells: spindle-shaped cells and large cuboidal or flattened cells. Here we have extended our previous observations (25) to demonstrate that the colonies also contain extremely small cells that are rapidly self-renewing (RS cells). The RS cells appear to be the earliest progenitors in the cultures and have the greatest potential for multilineage differentiation. They differ from more mature cells in the same cultures by a series of surface epitopes and expressed proteins. MethodsIsolation and Growth of MSCs. To isolate human MSCs, bone marrow aspirates of 10-20 ml were taken from the iliac crest of normal donors ranging in age from 19 to 49 years old under an Institutional Review Board approved protocol. Nucleated cells were isolated with a density gradient [Ficoll͞Paque (Pharmacia)] and resuspended in complete culture medium [␣ MEM, GIBCO͞BRL; 20% FBS, lot-selected for rapid growth of MSCs (Atlanta Biologicals); 100 units/ml of penicillin͞100 g/ml of streptomycin͞2 mM L-glutamine (GIBCO͞BRL)]. All of the cells were plated in 25 ml of medium in a 175-cm 2 culture dish (Falcon) and incubated at 37°C with 5% humidified CO 2 . After 24 h, nonadherent cells were discarded, and adherent cells ...

Effects of plating density and culture time on bone marrow stromal cell characteristics

“…In accordance with previous studies (15,16,19,20), two major subpopulations were defined. (a) MSC with a spindle shape had two processes extending in opposite directions from an elongated cell body.…”

“…(a) MSC with a spindle shape had two processes extending in opposite directions from an elongated cell body. This morphology has been termed immature, multipotent, RS1, and RS2 by others (15,16,20). (b) Large cells with a flattened, polygonal shape and a plainly visible nucleus were devoid of processes, or had many short processes.…”

“…For this expansion experiment, cells were replated at the appropriate seeding density (20,200 or 2000 cells/cm 2 ) at each passage. Cells were always passaged at 50% confluency, which was an average of every 10.8 days for MSC plated at 20 cells/cm 2 , 5.5 days for cells plated at 200 cells/cm 2 and 3.1 days for cells plated at 2000 cells/cm 2 .…”

“…It is obvious that these cultures are composed of different cell types with distinct morphologies. Attempts have been made to classify these subpopulations by marker expression and by shape and growth kinetics, e.g., as spindle-shaped type I or flattened type II cells (19) or as rapidly self-renewing cells, or mature MSC (15,16,20). In addition to the observed heterogeneity in vitro, variations have been observed in MSC cultures obtained from different species as well as from different rodent strains or individual human donors (21)(22)(23).…”

Cellules souches mésenchymateuses

“…Après la première phase de culture, les cellules doivent être détachées et réensemencées dans un nouveau flacon (passage) et la densité d'ensemencement doit être fortement diminuée [11]. Le choix de la densité cellulaire d'ensemencement est un paramètre crucial ; l'utilisation d'une faible voire très faible densité d'ensemencement pourrait permettre de maintenir un fort taux d'expansion et la multipotentialité des CSM cultivées [12]. Pour la production à usage clinique, l'utilisation d'une densité très faible est difficile car elle nécessite de très grandes surfaces de culture.…”