Proc. Natl. Acad. Sci. USA Vol. 92, pp. 1570-1574, February 1995 Immunology Long-term culture system for selective growth of human B-cell progenitors (cord blood/gene expression/interleukin 7/stem cell factor/murine stroma) DAVID J. RAWLINGS*, SHIRLEY G. QuANt, ROBERTA M. KATO*, AND OWEN N. WiTrE* *Department of Microbiology and Molecular Genetics, and tHoward Hughes Medical Institute, University of California, 5-748 MacDonald Research Laboratory, 10833 Le Conte Avenue, Los Angeles, CA 90024-1662 Communicated by Max D. Cooper, University ofAlabama, Birmingham, AL, October 21, 1994 ABSTRACT We describe a simple reproducible system for Establishment and maintenance of murine long-term cultures enrichment and long-term culture of human B-cell progeni- is dependent upon an adherent stromal cell microenvironment tors. Enriched CD34- cord blood mononudear cells are (4-6). Cultured cells develop and proliferate in direct association seeded onto a murine stromal cell line to establish a biphasic with stromal cells. While growth factors including interleukin 7 culture system. These cultures are characterized by transient (IL-7) and stem cell factor (SCF) are important for early B-cell growth of myeloid cells followed by outgrowth of cells highly lineage expansion, additional as-yet-unidentified stromal factors enriched for early B-cell progenitors. Cultures consisting of probably play a critical role in these processes (4, 5). Human >90%o early B-lineage cells [expressing CD10, CD19, CD38, B-lymphopoiesis is also likely to require close association with a and CD45 but lacking CD20, CD22, CD23, and surface IgM] stromal microenvironment. Human bone marrow stromal cells are maintained for > 12 weeks without growth factor addition. have been used to support short-term growth and proliferation of Cells remain predominantly germ line at the immunoglobulin CD10+, sIgM- human B-lineage cells (12, 14-16, 19). Establish- locus and express only low levels of cytoplasmic ,u chain, ment of long-term B-lymphoid cultures using human stroma has terminal deoxynucleotidyltransferase, and recombination- been limited in part by the need to repeatedly establish primary activating gene 1 product. They are unresponsive to the pre- stromal cultures and problems associated with transformed lines B-cell growth factors interleukin 7 or stem cell factor, or both, (20, 21). suggesting that growth support is provided by a cross-reactive Recently, several murine stromal lines have allowed support murine stromal cell factor. Cultured B-cell progenitors are of human hematopoietic progenitor cells in long-term cultures generated in large numbers (>108 cells from a typical cord and colony assays (22-24). Most notably, Baum et al. (24) blood specimen) suitable for use in biochemical analysis and established long-term cultures of Thy-1+, CD34+ cells on gene-transfer studies. This system should be useful for study murine stroma without additional growth factors. Those cells of normal and abnormal early human B-lymphopoiesis. remain capable of both myeloid and B-cell engraftment of in- tact human bone marrow implanted in severe combined im- Antigen-independent early B-cell development (B-lymphopoi- munodeficient (SCID) mice. In this paper we describe adap- esis) is characterized by an orderly expression of regulatory tation of these observations and conditions previously devel- genes, surface molecules, and gene rearrangements leading to oped for culture of murine B-cell progenitors to establish a the generation of surface IgM+ (sIgM+), sIgD+ mature B cells. long-term culture system for selective growth of human B-cell This process is controlled through cell-cell interactions and progenitors. soluble or cell-associated hematopoietic growth factors (1-3). B-lymphopoiesis occurs first in the fetal liver and bone marrow and continues after birth only in the bone marrow. The com- METHODS plexity of mammalian bone marrow precludes detailed in vivo Cord Blood Enrichment for CD34+ Cells, Culture Condi- analysis of these developmental events and has been signifi- tions, and Flow Cytometry. Heparinized (10 units/ml) cord cantly advanced through the establishment of long term B- blood was obtained from umbilical and placental tissues sched- lymphoid culture systems (4-10). Murine lymphoid progenitor uled to be discarded and stored up to 24 hr at room temper- cultures maintain murine immunoglobulin-locus-unrear- ature. Low-density mononuclear cells were collected by Ficoll/ ranged pro-B-cells that undergo limited differentiation. Mu- Hypaque separation (Pharmacia), and enrichment of CD34+ rine long-term bone marrow cultures support growth and cells was performed with a Celprate LC34 Biotin cell separa- spontaneous maturation of pre-B-cells into sIgM+ cells. How- tion column (CellPro, Bothell, WA) as recommended by the ever, cultured cells from both systems can fully reconstitute manufacturer. Briefly, cord mononuclear cells were stained B-cell function in immunodeficient animals (8, 9, 11). with anti-CD34 biotin-conjugated antibody and passed Several culture models have been useful in phenotypic and through an avidin column. Collected absorbed cells were 40- molecular analysis and in defining the growth factor require- 80% CD34+ by flow cytometry and represented approximately ments of early human B-cell progenitors (12-19). In these 0.5-1% of input cells. Sixty percent confluent S17 stromal cell systems, cultured B-cell progenitors survive for relatively short monolayers (25) were established 1-2 days prior to addition of periods (<4 weeks), and cell expansion is limited (usually CD34+ cord mononuclear cells by plating 2.5 x 105 S17 cells <10-fold). Isolation of highly purified human bone marrow per 10-cm2 tissue culture dish (Falcon). CD34+-enriched cells stem or B-cell progenitor populations and use of growth were plated at a density of 0.5-1 x 105 cells per ml onto the factors has been required to maintain optimal cell growth and S17-coated dishes (10 ml) in RPMI 1640 medium (Irvine expansion. Studies of early human B-lymphopoiesis would be Sciences) supplemented with 3% (vol/vol) defined fetal calf significantly advanced by the development of long-term cul- serum (HyClone) and 50 ,uM 2-mercaptoethanol and 200 mM ture models similar to those available using murine cells. Abbreviations: SCF, stem cell factor; IL-2 and IL-7, interleukins 2 and The publication costs of this article were defrayed in part by page charge 7; sIgM, etc., surface IgM, etc.; RAG1, recombination-activating gene payment. This article must therefore be hereby marked "advertisement" in 1; TdT, terminal deoxynucleotidyltransferase; C, constant region. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 1570 Downloaded by guest on September 30, 2021 Immunology: Rawlings et aL Proc. NatL Acad Sci USA 92 (1995) 1571 glutamine. Single-cell suspensions of cultured cells were incu- then mononuclear cells were enriched for CD34+ progenitor bated on ice for 30 min in saturating amounts of human- cells by using a CD34+ biotin avidin column. This enrichment specific monoclonal antibodies (Becton Dickinson, Southern step resulted in a mononuclear cell population containing Biotechnology Associates) in phosphate-buffered saline with between 50% and 80% CD34+ cells (average, =60% in four 2% calf serum, washed, and fixed in 1% paraformaldehyde. analyses), and further lineage depletion or CD34+ cell enrich- Samples were simultaneously analyzed by using isotype con- ment was not performed. Finally, CD34+-enriched cells were trols. Two-color flow cytometry was performed in a FACScan transferred onto 12-well or 10-cm2 tissue culture dishes con- and analyzed using Lysis II software (Becton Dickinson). The taining a subconfluent monolayer of the murine bone marrow fluorokine human IL-7-conjugated biotin and SCF-conjugated stromal cell line S17. The S17 stromal cell line was chosen biotin (R & D Systems) were used to detect IL-7 and SCF because of its ability to maintain long-term murine pro-B-cell receptors. Immunofluorescent terminal deoxynucleotidyl- growth (9, 10, 25, 30, 31). A seeding density of 0.1-5 x 105 cells transferase (TdT) staining (Supertechs, Bethesda, MD) was per ml resulted in the most consistent establishment of cul- performed on cytospin preparations. tures. Attempts to establish cultures by using unfractionated or Nucleic Acid Analysis. High molecular weight DNA and adherence-depleted cord mononuclear cells were unsuccess- total RNA were prepared and analyzed by standard methods. ful. For analyses, cultured cells were harvested with the ad- The human JH DNA probe (Oncogene Science) and other herent-cell monolayer by vigorous pipetting ahd passage probes were labeled by random primer DNA labeling (Primelt, through a needle and or cell sieve (40 ,um) to eliminate cell Stratagene) and had specific activities of 109 cpm/,ug of clumps. S17 cells could be easily distinguished from long-term DNA. cultured cells. Cytokines and Growth Factor Stimulation. Recombinant Establishment of long-term cultures followed three rela- human SCF was provided by K Zsebo and I. McNiece (Am- tively distinct phases: (i) acellular period-during weeks 1-2, gen). Recombinant human IL-7 and anti-IL-7 monoclonal cultures contained only small numbers of nonadherent cells antibody were purchased from Collaborative Biomedical and and significant cell death was observed; (ii) myeloid expan- Genzyme, respectively. For [3H]thymidine incorporation as- sion-during weeks 2-3, there was a marked increase in cel- says, 105 cultured cells per well were grown in medium de- lularity (up to 5 x 105 cells per ml) consisting primarily of scribed above or in S17-conditioned medium in the presence nonadherent myeloid cells identified by Wright stain and flow of growth factor for 48 hr and then were pulsed with 0.5 ,uCi cytometry; and (iii) outgrowth of B-cell progenitors- (1 ,uCi = 37 kBq) of [3H]thymidine for 18 hr before scintilla- beginning at 3-4 weeks, there was a decline in nonadherent tion counting with a multiwell harvester. (myeloid) cells and progressive outgrowth of small round cells tightly adherent to and burrowed within the stroma.