In Vitro Differentiation of Murine Sca-1 + Lin Cells into Myeloid, B Cell and T Cell Lineages

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1 In Vitro Differentiation of Murine Sca-1 + Lin Cells into Myeloid, B Cell and T Cell Lineages Maki Ito, Kazuaki Anan, Mahito Misawa, Shunrou Kai, Hiroshi Hara Department of Transfusion Medicine, Hyogo College of Medicine, Nishinomiya City, Japan Key Words. T cell differentiation Hematopoietic progenitors B cell differentiation Abstract. Hematopoietic progenitor cells were shown to be capable of differentiating into myeloid, B cell and T cell lineages. We used a two-step culture system in which enriched murine hematopoietic progenitors in bone marrow were first plated in viscid culture medium containing methylcellulose, erythropoietin (EPO), stem cell factor (SCF) and interleukin (IL)-7. One thousand enriched murine marrow cells formed 53.5 ± 12.1 (mean ± SD) primary colonies. Cells from a single blast colony were separated into two aliquots and replated in secondary methylcellulose cultures containing SCF and IL-7 for B cell lineage and SCF, IL-3, G-CSF, GM-CSF and EPO for myeloid lineage. Next, cells from five to ten primary blast colonies were cultured again in embryonal thymus (25 Gy irradiated). One aliquot of blast colonies in a primary culture contained four colony forming units (CFU) of granulocytes, erythroblasts, macrophages and megakaryocytes, eight CFU-granulocytes and macrophages, and 28 BFU-E in a representative secondary myeloid culture. Another aliquot formed a few B cell colonies (2-10) in a secondary B cell culture. B lymphoid colonies were composed of blast-like cells with B-220 antigen. T cells in a secondary T cell culture consisted of 16% L3T4 +, 16% CD8 + and 11% CD3 + of bone marrow origin in the thymus. From these results, we concluded that cells in the primary colonies from Sca-1 + Lin hematopoietic stem cells could differentiate into B cell, T cell and myeloid lineages. Stem Cells 1996;14: Introduction Hematopoietic stem cells (HSCs) are defined by their capacity for extensive self-renewal and their potential to differentiate into multiple cell Correspondence: Dr. Hiroshi Hara, Department of Transfusion Medicine, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya City, Hyogo, 663, Japan. Received October 10, 1995; provisionally accepted November 3, 1995; accepted for publication March 20, AlphaMed Press /96/$5.00/0 types one of the most remarkable and mysterious processes in developmental biology. In the fetus, HSCs are located in the liver, while in adult mammals, they are found in the bone marrow where they make up fewer than 1% of the cells. As HSCs do not show outstanding morphological characteristics, they are mostly studied indirectly by examination of their offspring. The importance of HSCs in the reconstitution of hemopoiesis by bone marrow transplantation prompted research aimed at stem cell isolation from more than 20 years ago. Although HSCs give rise individually to cells in multipotent lineages in vivo, no known HSCs can differentiate into all types of blood cells in vitro. Many researchers have reported cases of individual differentiation: T cell differentiation by Toki and Ikehara [1], the existence of bipotential precursors of B cells and myeloid lineage by Ogawa et al. [2], and the existence of bipotential precursors of B cells and macrophages by Cumano et al. [3]. We show here that HSCs can differentiate into B cell, T cell and myeloid lineages using viscid culture methods [1] and a cocultured method with stromal feeder layers [4]. In this paper, we describe Sca-1 + Lin cells that differentiated into T cell, B cell and myeloid lineages. Materials and Methods Mice C57BL/6J (H-2D b ) male mice (8 to 12 weeks) and BALB/c male and female adult mice (H-2D d ) were purchased from Japan Clea, Inc., Osaka, Japan. The mice were bred and maintained in our animal facilities. To obtain embryonal thymuses, male and female BALB/c mice were mated overnight, and females were examined the next morning. The day on which a vaginal plug was observed was considered day 0 of gestation. STEM CELLS 1996;14:

2 Ito, Anan, Misawa et al. 413 Reagents and Monoclonal Antibodies The culture mixture consisted of α-modified Eagle s medium (α-medium) (Flow; Irvine, Scotland), 1.2% (wt/vol) 1500 Centipoise methylcellulose (MeC) (Shinetsu Kagaku; Tokyo, Japan), 30% (vol/vol) fetal calf serum (FCS) (HyClone; Logan, UT), 1% bovine serum albumin (BSA) (Fraction V, Intergen; Purchase, NY), 0.1 mm 2-mercaptoethanol (2-ME) (Sigma; St. Louis, MO), and a combination of growth factors. Rat antimouse Ly2 (CD8a/53-6.7) monoclonal antibody (mab), rat antimouse granulocytes mab (Gr-1/RB6-8C5) [5], hamster antimouse CD3-ε/145-2C11 mab, rat antimouse Ly-5 (CD45R/B220) [6] mab, fluorescein-conjugated rat antimouse Ly-6A/E (Sca-1/D7) [7-9] mab, R phycoerythrin (R-PE) conjugated hamster antimouse CD3-ε and R-PE-conjugated rat antimouse CD-8a were obtained from Farmingen (San Diego, CA). Antimouse L3/T4 (CD4/YTS191.1) mab, mabto-mouse macrophage (MAC-1/M1/70.15) [10], mab-to-mouse B cells (CD45R, B220/RA-6B2) conjugated with fluorescein isothiocyanate (FITC) and anti-l3/t4 mab conjugated with R-PE CD4 were obtained from Cedarlane (Ontario, Canada). Dynabeads M-450 (magnetic beads coated with sheep antirat IgG) were obtained from Dynal (Oslo, Norway). Antimouse erythrocyte antigen mab, TER119 [11], was kindly provided by Tatsuo Kina, Department of Immunology, Chest Disease Research Institute, Kyoto University, Kyoto, Japan. Antimouse H-2D b and antimouse H-2D d FITC-conjugated mab were obtained from Meiji Nyugyo (Tokyo, Japan) and 5-fluorouracil (5-FU) from Kyowa Hakko (Tokyo, Japan). Growth Factors Purified recombinant human (rhu) interleukin 7 (IL-7) was provided by Dr. Mike Widmer (Immunex; Seattle, WA). Purified rhu erythropoietin (EPO), G-CSF, murine stem cell factor (SCF), IL-3 and GM-CSF were obtained from Kirin-Brewery Laboratories (Maebashi, Saitama, Japan). HSCs Adult mice were sacrificed four days after i.p. injection of 5-FU at 150 mg/kg [12]. Bone marrow cells were harvested from tibiae and femurs by flushing out with a 27-gauge needle using 10 ml of α-medium, and cells with a density between and were collected by Percoll density-fractionation techniques [12]. To remove the more mature cells, the cells were incubated for 30 min on ice with rat mab L3T4, CD8, CD3, Gr-1, Mac-1, B-220 and TER119. Next, they were washed and the antibody-coated cells were incubated with magnetic beads (Dynabeads M-450) coated with sheep antirat IgG for 30 min on ice. Cells coated with Dynabeads were removed using a magnet. As CD3 mab are derived from hamster, the sheep antirat IgG is a polyclonal antibody and displays cross-reaction to hamster IgG. After this procedure, the cells obtained were termed the negative lineage (Lin ). These Lin cells were incubated with Ly-6A/E (Sca-1) [7-9] conjugated FITC for 30 min on ice and sorted for Ly-6A/E + cells using a FACStar PLUS cell sorter (Becton Dickinson; San Jose, CA) (Fig. 1). Primary Culture Sca-1 + Lin HSCs were collected with FACStar PLUS and called the enriched cells. One thousand enriched cells were plated in a 35-mm suspension culture dish (#1008 Falcon, Becton Dickinson Overseas; Tokyo, Japan) with 1 ml α-medium containing 1.2% MeC, 30% FCS, 1% BSA, 0.1 mm 2-ME, SCF (100 ng/ml), IL-7 (500 units/ml) and EPO (2 units/ml) [2]. Dishes were incubated at 37 C in a humidified atmosphere flushed with 5% CO 2 /95% air for seven days. Secondary Myeloid and B Cell Cultures The primary colony was lifted from the medium and divided into two aliquots which were then plated in secondary cultures to study myeloid or B cell potentials of the cells in primary colonies. The secondary myeloid culture was carried out in a 35-mm suspension culture dish with 1 ml α-medium containing 1.2% MeC, 30% FCS, 1% BSA, 0.1 mm 2-ME, SCF (100 ng/ml), IL-3 (20 ng/ml), G-CSF (10 ng/ml), GM-CSF(10 ng/ml) and EPO (2 units/ml). The secondary B cell culture was carried out in a 35-mm suspension culture dish in α-medium containing 30% FCS, 1% BSA, 0.1 mm 2-ME, SCF (100 ng/ml) and IL-7 (500 units/ml) [2]. Both cultures were incubated at 37 C in a humidified atmosphere flushed with 5% CO 2 /95% air for 7 to 10 days. B cell colonies in the secondary culture were stained with B-220 mab labeled with FITC after being picked up and analyzed

3 414 In Vitro Differentiation of Murine Sca-1 + Lin Cells Figure 1. A) Light scattering characteristics of Sca-1 + Lin bone marrow cells. The cells were analyzed for lightscattering characteristics. B) Histogram of Sca-1 + Lin bone marrow cells. The cells were gated and sorted using a FACStar PLUS cell sorter. by FACScan. Granulocyte/erythroid/macrophage/megakaryocyte (GEMM) colonies were confirmed by staining with May-Grünwald-Giemsa-staining solution and for acetylcholinesterase (AchE) after they had been picked up, in order to demonstrate the presence of megakaryocytes [4, 13]. Secondary T Cell Culture Five to ten primary colonies were lifted from the medium and placed in a thymic lobe (25 Gy irradiation). This organ culture system for studying the differentiation of T cells from HSCs has been described by Toki et al. [1]. Briefly, thymic cells from newborn mice were cultured with medium (3 ml α-medium/containing 10% FCS/50µM 2-ME) in the lower chamber (#3046 Falcon) as a feeder layer. A Nunc Tissue Culture Inserts chamber (pore size, 0.02 µm; diameter, 10 mm) (Nunc; Naperville, IL) was placed in the lower chamber. An embryonal (before gestation day 15) thymic lobe irradiated with 25 Gy was placed inside the upper chamber. One day later, cells from 5 to 10 colonies in 200 µl of α-medium were added to the upper chamber and cultured for two weeks in a CO 2 incubator with humidified 5% CO 2 /95% air at 37 C. The embryonal thymus taken from the upper chamber after incubation was crushed between slide glasses to obtain a single cell suspension. After washing, the cells were first incubated with FITC-conjugated-anti-H-2D b rat mab to detect cells derived from HSCs in bone marrow cells. Next, the cells were stained with PE-conjugated-anti-L3T4, CD8 and CD3 mab, and analyzed using a FACScan. Results Primary Colonies in Methylcellulose Culture The enriched primary culture cells (1000 cells/well) were plated in methylcellulose culture medium containing SCF, EPO and IL-7. A total of 53.5 ± 12.1 (mean ± SD) colonies in six experiments were formed from the enriched cells on day 7 of culture. A few of them, composed of neutrophils and macrophages, consisted of small cells and could be distinguished by cell size from the blast colonies. The remaining colonies consisted of blast cells with the potential for multilineages, i.e., for revealing neutrophils, eosinophils, macrophages, erythrocytes and megakaryocytes. All the primary blast colonies cultured in a secondary culture formed some myeloid colonies and some B cell colonies. Myeloid and B Cell Lineage in Secondary Culture A single primary blast colony was separated into two aliquots after being picked up with a Pasteur pipette. One aliquot was placed in a methylcellulose culture medium containing SCF, IL-3, G-CSF, GM-CSF and Epo (myeloid culture) [14]. Another aliquot was cultured in a methylcellulose culture medium containing SCF and IL-7 (B cell culture). After a seven-day incubation in

4 Ito, Anan, Misawa et al. 415 secondary culture, 16 colonies from colony-forming units granulocyte-macrophage (CFU-GM), 56 colonies from BFU-E, eight colonies from CFU-GEMM in myeloid culture and 8.8 B cell colonies in B cell culture (means of six experiments) from cells in a primary blast colony were counted in six experiments. All the primary blast colonies contained cells forming myeloid colonies and B cell colonies (Fig. 2). Morphologic examination of the Cytospin slide preparations with May-Grünwald-Giemsa solution revealed that the cells were composed of mature cells containing macrophages, granulocytes, erythrocytes and megakaryocytes. We studied them using the acetylcholinesterase staining method (Fig. 3) and confirmed the presence of megakaryocytes. The other aliquot was placed in methylcellulose culture medium containing SCF and IL-7. Although there were few B cell colonies in the secondary culture, they appeared to be similar to blast colonies. These cells were picked up and stained with FITC-conjugated B-220, then analyzed by FACScan (Fig. 4). Almost all cells in the colonies were B-220-positive. T Cell Lineage in Secondary Culture The cells in the embryonal thymus were lifted from the upper chamber after a two-week Figure 2. Number of BFU-E, CFU-GM and CFU- GEMM and B cell progenitors in a primary blast colony. A primary colony was lifted from the primary culture and divided into two aliquots. One aliquot was plated in secondary culture containing SCF, IL-3, GM-CSF, G-CSF and EPO. Another aliquot was plated in secondary culture containing SCF and IL-7. Dishes were incubated at 37 C in a humidified atmosphere flushed with 5% CO 2 /95% air for 7 to 10 days. Figure 3. Megakaryocyte examination of CFU- GEMM. Single primary blast colonies were suspended and separated into two aliquots. One aliquot was placed in a myeloid secondary culture. After a 14-day incubation, the colonies were counted. Colonies considered to be from CFU-GEMM were picked up and stained for acetylcholinesterase activity. A large number of acetylcholinesterase positive cells and many small negative cells were found in a part of the Cytospin preparations. incubation and checked for cellular elements. These cells were stained with FITC-conjugated H-2D b rat mab, cells not derived from the embryonal thymus cells, and PE-conjugated L3T4, CD8 or CD3 mab. Analysis of the cells using a flow cytometer revealed that 43.0 ± 6.4 (mean ± SD) (n = 6) cells from the embryonal thymus were derived from bone marrow Sca-1 + Lin HSCs. L3T4 + and CD8 + each accounted for approximately 16% of the cells originating from the bone marrow cells, respectively (Fig. 5). Although we found these cells from Sca-1 + Lin cells to be L3T4 + or CD8 +, we could not identify them as double-positive (L3T4 + and CD8 + ) or single-positive (L3T4 + or CD8 + ) cells. However, these cells originating from Sca-1 + Lin bone marrow cells definitely showed T cell markers. Discussion In the present study, we demonstrated the in vitro differentiation of adult murine bone marrow hematopoietic progenitors into both lymphoid and myeloid lineages. In vitro reconstitution

5 416 In Vitro Differentiation of Murine Sca-1 + Lin Cells Figure 4. FACScan analysis of B cell colony in secondary culture containing SCF and IL-7. A primary colony was lifted from the primary culture and divided into two aliquots. One aliquot was plated in secondary culture containing these cytokines. Dishes were incubated at 37 C in a humidified atmosphere flushed with 5% CO 2 /95% air for 7 to 10 days. B cell colonies were stained with B-220 mab after being picked up and analyzed by FACScan. Control 1: Cells from a B cell colony were stained without any mab. Control 2 and 3: Cells from a B colony were stained with H-2D d mab or H-2D b mab as a negative and positive control, respectively. of hematopoiesis has been extensively studied and, in fact, the differentiation into individual lineages has been demonstrated. Observation of bone marrow transplantation recipients who develop all of the lineages clearly indicates that multipotent stem cells do reside in adult bone marrow and can differentiate into all lineages under appropriate conditions. However, this is the first report of HSCs differentiating into B cell, T cell and myeloid lineages in vitro. We introduced a two-step culture method to analyze the differentiation of stem cells into the individual lineages in appropriate environments, and successfully demonstrated the multipotency of adult bone marrow hematopoietic progenitors in vitro. The starting material we selected was Sca-1 + Lin cells. This selection is very important because mouse hematopoietic stem cells were shown to express Sca-1 antigen [15]. Also, we provided media with combinations of cytokines to optimize the culture conditions because the requirements of hematopoietic progenitors presumably differ with each step and lineage. A combination of SCF and IL-7 in the secondary culture is considered to be optimal for the differentiation into B cells [2], while a combination of SCF, IL-3, GM-CSF, G-CSF and EPO in the secondary culture is considered to be appropriate for the differentiation into myeloid lineages [2, 4, 9, 12]. The most important point of our work was the demonstration of T cell lineage differentiation because it is considered to require a thymic microenvironment [15-17]. There are no clear answers yet to questions concerning which cell population or what kinds of humoral factors [14, 18-20] are essential to T cell development. In some studies, T cell differentiation was demonstrated in vivo by transferring the progenitor cells into severe combined immunodeficiency (SCID) mice. We used thymic cells from newborns and irradiated the fetal thymic lobe from H-2D mismatched mice to distinguish cell origin. In bone marrow transplantation recipients, the T cell appears later than in other lineages. Previous observations on T cell differentiation of the fetal liver hematopoietic stem cell in a fetal thymic organ culture system [21] and intrathymic T cell differentiation of purified stem cells in lethally irradiated mice [22] show that the increase of T cells follows the maximal number of cells in myeloid lineages. These observations raise the question of whether the development of T cells in the thymus requires prior generation of myeloid lineage. In our study, the existence of viable myeloid cells was excluded. However, the possibility remains that a humoral environment created by preexisting cells in the thymus influenced the T cell development. Weissman et al. indicated that Sca-1 + Thy low Lin cells are murine HSCs [15]. However, we could not select them with Thy-1 mab because our FACStar PLUS has only one laser. Sca-1 + Lin cells might be committed to T cells because they contain Thy-1 high cells [9, 22-26]. Analysis of the primary colonies in the first culture containing SCF, IL-7 and EPO revealed that the T cell element was not detectable (data not shown). These results indicate that Sca-1 + Thy-1 high Lin cells could not proliferate in the primary colonies under these conditions.

6 Ito, Anan, Misawa et al. 417 Figure 5. FACScan analysis of T cells in secondary culture. Five to ten primary colonies were lifted from the primary culture and placed in an embryonal thymic lobe (25 Gy irradiation). These cocultured colonies were incubated at 37 C in a humidified atmosphere flushed with 5% CO 2 /95% air for two weeks. Thymus was lifted from secondary culture, and cells obtained from it were first stained with FITC-H-2D b, then with PE-L3T4, CD8 and CD3. Myeloid differentiation of human fetal liver HSCs has also been demonstrated in human thymic organ culture [21]. We could not show myeloid differentiation of the progenitors in the coculture with thymus. However, we cannot exclude the possibility that the potential of differentiation of fetal liver cells is different from that of adult bone marrow cells. Assay methods to verify the differentiation to each lineage were also critical to our study. Primary colonies arising in the first culture step included colonies of blast-like cells under microscopic observation. We picked up these colonies, separated them into two aliquots, and put them individually into the secondary myeloid and B cell cultures. For evaluation of myeloid differentiation, we morphologically examined cells in colonies of the secondary myeloid culture. The staining techniques used revealed the presence of mature myeloid cells. For evaluation of B cells, we analyzed the blast-like colonies in the secondary B cell culture by FACScan using B-220 mab. All blast colonies in the secondary myeloid and B cell cultures formed some myeloid colonies and some B cell colonies. Therefore, B cells and cells in all the myeloid lineages were considered to have been derived from a single colony, which means they were derived from a single progenitor clone. On the other hand, T cells do not form colonies in vitro. We examined the presence of T cells in several secondary B cell colonies by FACScan using L3T4, CD8 and CD3 mab. Although we could not specify the T cells in a single secondary colony, we could show the T cell lineage differentiation of the progenitors in the primary blast colonies that could form B cell myeloid colonies. Since all the primary blast colonies contained cells that could form B cell colonies and/or myeloid colonies, the present results indicate that the colonies contained cells having the ability of T cell differentiation. We were successful in demonstrating the in vitro differentiation of adult bone marrow hematopoietic progenitors into the full line-up of lineages. This should aid efforts to improve the

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