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EMBRYONIC STEM CELLS

Direct Development of Functionally Mature Tryptase/Chymase Double-Positive Connective Tissue-Type Mast Cells from Primate Embryonic Stem Cells

Departments of ^aPediatrics and
^cDermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan;
^bDivision of Cellular Therapy, Institute of Medical Science, University of Tokyo, Tokyo, Japan;
^dResearch Center for Regenerative Medicine, Kyoto University, Kyoto, Japan;
^eResearch Center for Animal Life Science, Shiga University of Medical Science, Ohtsu, Japan;
^fDepartment of Dermatology, Chiba University Graduate School of Medicine, Chiba, Japan

Key Words. Embryonic stem cells • Mast cells • Primate • Development • Chymase • Tryptase

Correspondence: Correspondence: Tatsutoshi Nakahata, M.D., Ph.D., Department of Pediatrics, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Telephone: 81-75-751-3290; Fax: 81-75-752-2361; e-mail: tnakaha{at}kuhp.kyoto-u.ac.jp

Received on May 8, 2007; accepted for publication on October 25, 2007.

First published online in STEM CELLS EXPRESS  November 8, 2007.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

 
Conditions that influence the selective development or recruitment of connective tissue-type and mucosal-type mast cells (MCs) are not well understood. Here, we report that cynomolgus monkey embryonic stem (ES) cells cocultured with the murine aorta-gonad-mesonephros-derived stromal cell line AGM-S1 differentiated into cobblestone (CS)-like cells by day 10–15. When replated onto fresh AGM-S1 with the addition of stem cell factor, interleukin-6, and Flt3 ligand, these CS-like cells displayed robust growth and generated almost 100% tryptase/chymase double-positive MCs within 3 weeks. At all time points, the percentage of tryptase-positive cells did not exceed that of chymase-positive cells. These ES-derived MCs were CD45+/Kit+/CD31+/CD203c+/HLA-DR– and coexpressed a high-affinity IgE receptor on their surface, which was upregulated after IgE exposure. Electron microscopy showed that they contained many electron dense granules. Moreover, ES-derived MCs responded to stimulation by via IgE and substance P by releasing histamine. These results indicate that ES-derived MCs have the phenotype of functionally mature connective tissue-type MCs. The rapid maturation of ES-derived MCs suggests a unique embryonic pathway in primates for early development of connective tissue-type MCs, which may be independent from the developmental pathway of mucosal-type MCs.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

 
Multipotential progenitor cells, capable of becoming mast cells (MCs), leave the bone marrow (BM) and enter the circulation, but they complete their differentiation into mature MCs only after arriving in peripheral tissues [1]. Mature MCs can be distinguished from other cell types by surface expression of the high-affinity IgE receptor (FcRI), high levels of c-Kit, and characteristic secretory granules. In addition to histamine, MC granules contain many other neutral proteases, such as tryptase, chymase, carboxypeptidase and cathepsin G. Predicted biologic outcomes of tryptase might include anticoagulation, fibrosis and fibrolysis, kinin generation and destruction, cell surface protease activated receptor (PAR)-2 activation, enhancement of vasopermeability, angiogenesis, inflammation, and airway smooth muscle hyperreactivity. Chymase is a potent activator of angiotensin I, inactivates bradykinin and PAR-1 receptors, attacks the basement membrane of skin, processes type I procollagen, and stimulates mucus production from glandular cells. Biologically active c-Kit can be released from the cell surface by chymase. Although tryptase and chymase serve as the selective markers that distinguish MCs from other cell types and different MC subpopulations from one another, physiological functions for these enzymes in MCs are still not well-defined. In humans, two types of MCs have been identified based on their neutral protease compositions [2]. Connective tissue-type MCs (CT-MCs), predominantly located in normal skin and in intestinal submucosa, contain tryptase, chymase, MC carboxypeptidase, and cathepsin G in their secretory granules and are therefore named MC_TC. Mucosal-type MCs (M-MCs), the main type of MCs in normal alveolar wall and in small intestinal mucosa, contain tryptase in their secretory granules but lack the other proteases, and are named MC_T.

Conditions that influence the selective development or recruitment of CT-MCs and M-MCs are not well understood. Stem cell factor (SCF)-dependent in vitro-derived human MCs are considered as a model of M-MCs because they have a low percentage of chymase-expressing cells and display M-MC type immunopharmacological responses [3]. In these cultured MCs, cytokines such as interleukin (IL)-4, IL-6, and nerve growth factor reportedly induce chymase expression [4–6], but these chymase-positive cells do not release histamine in response to stimulation with substance P. Additional in vivo observations suggest that CT-MCs and M-MCs develop along distinct pathways. In humans with inherited combined immunodeficiency disease or with AIDS, marked and selective decreases in M-MC concentrations occur in the bowel, whereas the concentration and distribution of CT-MCs are unaffected [7]. This suggests that the appearance of M-MCs in tissues is dependent on functional T lymphocytes and that CT-MC development proceeds independently.

In murine fetal stages, MC precursors are highly concentrated in the yolk sac and fetal blood, suggesting that there exists a strong early embryonic wave of MC development [8, 9]. However, because of the impossibility of conducting experimental manipulations on human embryos, little is known about human MC development during the embryonic and fetal stages. Old World monkeys, such as the cynomolgus monkey (Macaca fascicularis), are widely used for medical research [10]. Recently, primate embryonic stem (ES) cell lines have been established, and hematopoietic differentiation from primate ES cells was induced successfully in vitro [11, 12]. Therefore, monkey ES cells may provide a useful model to elucidate early hematopoietic development in nonhuman primates as well as in human.

Here, we report the development of MCs from cynomolgus monkey ES cells by coculture with the murine aorta-gonad-mesonephros (AGM)-derived stromal cell line AGM-S1 with the addition of SCF, IL-6, and Flt3 ligand (FL). The primate ES-derived MCs contain abundant, metachromatically stained granules and were positive for both tryptase and chymase from early time points in their development. They express the high-affinity IgE receptor (FcRI) on their surface, and the FcRI is upregulated after exposure to IgE. Moreover, the ES-derived MCs responded to stimulation with IgE and substance P by releasing histamine. These results indicate that the primate ES-derived MCs have the phenotype of CT-MCs. The rapid maturation of ES-derived MCs suggests a unique embryonic pathway in primates for the early development of CT-MCs, which may be independent from the M-MC pathways.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

 
Cell Lines
The cynomolgus monkey ES cell line (CMK6), which had been transfected with an enhanced green fluorescent protein (GFP) driven by CAG promoter using Lipofectamine 2000 [13], was maintained as described [11]. The establishment of AGM-S1 cells and their potential to support human hematopoiesis had been reported elsewhere [14]. AGM-S1 cells within 10 passages were used throughout our experiments. AGM-S1 cells (1–2 x 10⁵) were cultured in 35-mm gelatin-coated dishes overnight to make a confluent feeder layer. On the next day, they were exposed to 15 Gy of radiation. The human MC line HMC-1 was cultured in -minimum essential medium (-MEM) containing 10% fetal bovine serum (FBS). The procedures using primate ES cells, experimental animals, and human samples in this study were approved by the Internal Review Board of Kyoto University.

Coculture of Primate ES Cells with Murine AGM-S1 Cells
To induce hematopoietic differentiation, we used a primate ES cell and murine AGM-S1 cell coculture system that is similar to that in our recent report on human ES cell and murine fetal liver stromal cell coculture [15]. Briefly, approximately 20–30 undifferentiated primate ES cell colonies (each consisting of 500–1,000 cells) were physically picked up under a microscope using a fine glass rod, transferred in each well of six-well plate (diameter, 35 mm) that had been covered with irradiated confluent AGM-S1 cells, and cultured in -MEM containing 15% FBS and no added cytokines. Culture medium was replaced every 3 days. At given time points, whole cells were collected with a 0.05% trypsin/EDTA solution (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) for further use.

Hematopoietic Colony Assay
Hematopoietic colony assay was done as described previously [16]. Briefly, 1 ml aliquots of a culture mixture containing primate ES/AGM-S1 coculture cells or ES-derived day 12–15 cultured cells, -MEM, 1.2% methylcellulose (Shinetsu Chemical, Tokyo, http://www.shinetsu.co.jp), 30% FBS, 1% deionized fraction V bovine serum albumin (BSA), 10^–4 M mercaptoethanol (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), and a cocktail of cytokines with SCF, IL-6, IL-3, granulocyte-colony stimulating factor, thrombopoietin, and erythropoietin were plated in 35-mm suspension culture dishes (Nunc, Naperville, IL, http://www.nuncbrand.com). Colony types were determined on days 7–14 of the incubation by in situ observations using an inverted microscope, according to criteria established by Nakahata and Ogawa [17].

Harvest of Primate ES-Derived Cells and Secondary MC Cultures
Using a 10-µl microtip (Molecular BioProducts, San Diego, http://www.mbpinc.com/html/index.html), 5,000–10,000 primate ES-derived cobblestone (CS)-like cells were physically picked up on days 12–15 of the coculture and replated into a 35-mm culture well containing a freshly prepared, irradiated AGM-S1 feeder layer in medium containing 15% FBS with SCF, IL-6, and FL (secondary MC culture). Cytokines and their doses were as follows: 100 ng/ml SCF, 100 ng/ml IL-6 (both provided by Kirin Brewery, Maebashi, Japan, http://www.kirin.co.jp/english), and 20 ng/ml FL (R&D Systems Inc., Minneapolis, http://www.rndsystems.com). Half of the volume of culture medium was replaced with fresh medium twice a week.

Morphological Observations and Immunochemistry
From day 12 on, cells in secondary MC cultures were picked up and centrifuged onto glass slides every 3–6 days. Centrifuged preparations were then stained with May-Grünwald-Giemsa, acidic toluidine blue, Alcian Blue, and Safranin O solutions. For MC-specific tryptase and chymase assays, an alkaline phosphatase anti-alkaline phosphatase (APAAP) method (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com) was used according to the manufacturer's instructions [16], and anti-human tryptase monoclonal antibody (mAb) (DakoCytomation) and anti-human chymase mAb (Chemicon, Temecula, CA, http://www.chemicon.com) were used. In some experiments, frozen cynomolgus monkey skin samples were stained by the same APAAP method to detect tryptase- and chymase-containing skin MCs.

Flow Cytometry
For immunological staining, cultured cells were preincubated with 10 µl of normal rabbit serum for 30 minutes to block Fc receptors on the cell surface. After a wash by phosphate-buffered saline (PBS) with 0.1% BSA, cells were stained for 30 minutes on ice with various mAbs conjugated with phycoerythrin (PE) or allophycocyanin (APC) or unconjugated mAbs. Samples stained with unconjugated mAbs were then incubated with PE- or APC-conjugated goat-derived anti-mouse mAbs for an additional 30 minutes. Stained cells were washed with BSA-containing PBS and analyzed using a FACSCalibur cytometry system (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) with CellQuest software (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Propidium iodide-stained dead cells were gated out. mAbs against CD34, CD31, and HLA-DR (BD Biosciences), CD45 (DakoCytomation), c-Kit (Nichirei, Tokyo, http://www.nichirei.co.jp/english/index.html), FcRI (CRA-1; eBioscience Inc., San Diego, http://www.ebioscience.com), and CD203c (Beckman Coulter, Miami, FL, http://www.beckmancoulter.com) were used. Isotopes of mouse IgG (BD Biosciences) and goat IgG (DakoCytomation) were used as the negative controls.

Reverse Transcription-Polymerase Chain Reaction
To detect early development of the primate ES-derived hematopoiesis, total RNA was prepared for reverse transcription-polymerase chain reaction (RT-PCR) using TRIzol (Invitrogen) and then reverse-transcribed to cDNA using a SuperScript first-strand synthesis system for RT-PCR (Invitrogen). The oligonucleotide primers were as follows: HPRT, forward (F), 5'-CTTGCGACCTTGACCATCTTTGGA-3', and reverse (R), 5'-GGCGTCGTGATTAGTGATGATGAACC-3'; OCT-4, F, 5'-CGTTCTCTTTGGAAAGGTGTT-3', and R, 5'-ACACTCGGACCACGTCTTTC-3'; c-kit, F, 5'-CAAGATTAGAAGCTGAAAACCT-3', and R, 5'-TCAAATCCATTGAGTACAATGC-3'; GATA1, F, 5'-GATCCCACAACTACATGGAAC-3', and R, 5'-ACAGTTGAGCAATGGGTACACC-3'; and CD34, F, 5'-TGCACCCTGTGTCTCAACATGG-3', and R, 5'-GCACAGCTGGAGGTCTTATTTTGC-3'. Primers for -fetoprotein, Nestin, Brachyury, FLK-1, SCL, and GATA-2 have been published elsewhere [12].

Since the cynomolgus monkey-specific sequences were not available, we used the corresponding human or other Old World monkey genes to design our PCR primers for detection of MC-specific tryptase and chymase: glyceraldehyde-3-phosphate dehydrogenase, F, 5'-CGGGAAGCTTGTCATCAATGG-3', and R, 5'-GGCAGTGATGGCATGGACTG-3'; tryptase, F, 5'-GGAGCAG-CACCTCTACTACC-3', and R, 5'-ATTCACCTTGCACACC-AGGG-3'; and chymase, F, 5'-AAGGAGAAAGCCAGCCTG-ACC-3', and R, 5'-TCCGACCGTCCATAGGATACG-3'. PCR-amplified samples were sequenced by the dideoxynucleotide termination method with an automated sequencer (ABI3100; Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com).

Activation of MCs
For IgE stimulation, day 40 primate ES-derived MCs were cultured with the addition of 4 µg/ml IgE in the medium for 3 days. Cells were then harvested, washed, and stained with mAb against human FcRI (CRA-1). In activation experiments, day 40 primate ES-derived MCs were washed and suspended in histamine-release buffer and preincubated for 5 minutes at 37°C. For stimulation, 5 µl of CRA-1 or control mouse IgG, or substance P (Sigma-Aldrich), was added to a 25-µl cell suspension (1 x 10⁶ cells per milliliter) and incubated for an additional 15 minutes at 37°C. The reaction was stopped by adding 200 µl of ice-cold buffer. The cells were separated by centrifugation at 300g for 7 minutes at 4°C, and the supernatant was collected. The cell pellet was resuspended in 200 µl of buffer containing 0.5% Triton X-100 and 0.1% BSA and quick frozen in liquid nitrogen and thawed four times. After centrifugation at 12,000g for 15 minutes at 4°C, the soluble extract was collected. Histamine levels were measured by an enzyme-linked immunosorbent assay (ELISA) (Beckman Coulter), and β-hexosaminidase activity was measured with pNA-β-D-glucosaminide in 0.1 M sodium citrate buffer, pH 4.5, as described previously [18].

Assay of Intracellular Histamine
The intracellular histamine concentration of cell lysates was measured using an ELISA histamine assay kit (Medical & Biological Laboratories, Nagoya, Japan, http://www.mbl.co.jp/e/index.html). Briefly, 500 ES-derived MCs were washed and pooled in 0.5 ml of PBS on days 40–50. The cell suspensions were then briefly frozen in liquid nitrogen and soon returned to a 37°C water bath to thaw. The freeze-thaw was repeated twice, and the cell lysates were then examined for histamine content according to the manufacturer's protocol. For comparison, histamine concentration of day 70 human cord blood (CB)-derived MCs were analyzed by the same method.

MC Culture from CB Progenitors
Human MC culture was performed as reported previously [19], with slight modifications. Briefly, human CB was obtained from a normal full-term delivery with informed consent. Mononuclear cells were separated by density-gradient centrifugation, and CD34+ cells were isolated using a CD34 progenitor cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). The separated cells were cultured with 5% FCS, 100 ng/ml SCF, and 50 ng/ml IL-6 in AIM-V medium (Invitrogen).

Statistical Analysis
In some experiments, data are presented as the mean ± SD. Statistical significance was determined with the Student t test. p values less than .05 were considered significant.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

 
Development of Multipotential Hematopoietic Progenitor Cells from Primate ES Cells
Primate ES cells, cocultured on a confluent monolayer of irradiated AGM-S1 cells [14] in -MEM containing 15% FCS and no added cytokines, began to differentiate concomitantly with rapid proliferation at day 3–5. By days 8–10, some areas where the cells accumulated grew to form translucent sac-like structures containing bright, small, round cells that looked like congested CS-like cells (Fig. 1A). Because we used cynomolgus monkey ES cells that were transfected with GFP, fluorescent microscopy revealed that these CS-like cells were positive for GFP, indicating that they were derived from the ES cells (Fig. 1B).

View larger version (85K): [in this window] [in a new window]   Figure 1. Cynomolgus monkey embryonic stem (ES)-derived cobblestone (CS)-like cells in coculture with AGM-S1 cells. (A): CS-like cells derived from ES cells in coculture with AGM-S1 cells on day 14 (original magnification, x200). (B): Fluorescence micrograph of (A) showing that CS-like cells are derived from green fluorescent protein-positive ES cells (original magnification, x200). (C): May-Grünwald-Giemsa staining of ES-derived day 14 CS-like cells (original magnification, x400). (D): ES-derived CS-like cells were positive for CD34, as indicated by anti-human CD34 APAAP staining (original magnification, x400). (E): May-Grünwald-Giemsa staining of undifferentiated ES cells. (F): Negative staining of CD34 by alkaline phosphatase anti-alkaline phosphatase (cells were only shown the counterstaining of undifferentiated ES cells by Carrazi's hematoxylin solution). (G): A fluorescence-activated cell sorting profile shows that ES-derived CS-like cells are almost all CD34 positive. NG shows the negative staining for CD34 on undifferentiated ES cells. Abbreviations: APC, allophycocyanin; NG, negative control.

At day 14, the CS-like cells in the transparent sac-like areas had proliferated to the extent that we were able to pick them up by a simple physical method. Routinely, a single 35-mm culture well gave rise to 1–2 x 10⁴ CS-like cells at days 10–15. These CS-like cells showed a homogeneous phenotype of mononucleated small cells, similar to undifferentiated hematopoietic stem/progenitor cells, as indicated by May-Grünwald-Giemsa staining (Fig. 1C). More than 95% of the primate ES-derived CS-like cells were CD34+ by immunochemical staining and fluorescence-activated cell sorting (FACS) analysis (Fig. 1D, 1G), whereas undifferentiated ES cells were negative for CD34 expression (Fig. 1E–1G). A clonal cell culture showed that hematopoietic activities were highly concentrated in these CS-like cells. ES-derived day 14 CS-like cells, when planted at 5,000 per assay, generated 234 ± 36 total hematopoietic colonies (including 24 ± 6 mixed colonies), which was 134-fold more than the total coculture cells at the same time point (supplemental online Table 1). These CS-like cells also generated CD144+/CD31+ endothelial-like cells when cultured by adding vascular endothelial growth factor (data not shown). Thus, the primate ES-derived CS-like cells may represent a highly concentrated fraction of hematopoietic and hemangioblastic progenitors.

MC Differentiation from ES-Derived CS-Like Cells
Physically harvested day 14 ES-derived CS-like cells were replated in a secondary coculture on newly irradiated AGM-S1 cells to induce MC differentiation (secondary MC culture) with a cytokine cocktail of SCF, IL-6, and FL. ES-derived phase-contrast dark cell clusters appeared within 24 hours (Fig. 2A). These cell clusters grew gradually during the first 5 days and showed a robust cell proliferation from day 7, with the phase-dark cells floating in the medium and becoming bright, round, small cells. These cells further proliferated during the following week and gradually developed granules in the cytoplasm (Fig. 2B). GFP expression demonstrated that these granule-containing cells were all derived from ES cells (Fig. 2C).

View larger version (149K): [in this window] [in a new window]   Figure 2. Phenotype of cynomolgus monkey embryonic stem (ES)-derived mast cells (MCs). (A): Phase-contrast dark cell clusters could be observed after 3 days when day 14 primary ES-derived cobblestone-like cells were replated on aorta-gonad-mesonephros-S1 cells in secondary MC cultures (original magnification, x100). (B): On day 12, secondary MC culture produced a uniform cell population of round, small, granule-containing cells (original magnification, x200). (C): Green fluorescent protein-expressing cells shown in (B) (original magnification, x200). (D): On day 21 of secondary MC culture, ES-derived cells displayed a typical MC phenotype when stained by May-Grünwald-Giemsa solution (original magnification, x400). (E): Cells from these cultures stained with toluidine blue (original magnification, x400). (F): The same cultures stained with Alcian Blue (original magnification, x400). (G): Safranin O-stained cells from the same cultures. (H): Electron micrograph of day 40 ES-derived MCs. Typical rough, dark granules; pseudopodia; highly developed mitochondria; and Golgi bodies could be observed. (I): An enlarged view of the box in (H), showing granules with various electron densities.

View larger version (39K): [in this window] [in a new window]   Figure 3. Fluorescence-activated cell sorting profiles of embryonic stem (ES)-derived day 21 mast cells (MCs). A homogeneous cell population of low side scatter and forward scatter could be seen (upper left panel), most of which expressed green fluorescent protein (middle left panel). The monkey ES-derived MCs were c-Kit+/CD45+/CD31+/CD203+/FcRIdim+/HLA-DR–.

View larger version (17K): [in this window] [in a new window]   Figure 4. Proliferation, recruitment, and cytokine requirements of cynomolgus monkey embryonic stem (ES)-derived mast cells (MCs). (A): 5 x 103 ES-derived cobblestone (CS)-like cells were plated in a 35-mm culture dish in secondary MC culture. At given times, floating cells were quantified. A peak of 480-fold proliferation could be observed at day 21. (B): Continuous proliferation of ES-derived MCs starting from day 40 of secondary MC culture to day 80, with 10-day replating intervals, showing typical characteristic of MC recruitment. (C): 1 x 104 ES-derived CS-like cells were replated in a 35-mm culture dish in the secondary MC culture with various cytokine combinations. On day 20, the number of floating cells was quantified. Results are shown in converted values from three different experiments, normalized to the highest cell growth of the three-cytokine combination (stem cell factor + interleukin-6 + Flt3 ligand) as 100%. The MC phenotype was confirmed by May-Grünwald-Giemsa staining. Abbreviations: FL, Flt3-ligand; IL-6, interleukin-6; SCF, stem cell factor.

ES-Derived MCs Are Positive for both Tryptase and Chymase
With mAbs reactive to human MC specific tryptase and chymase, we found that approximately 10% of ES-derived MC cells stained double-positively for both proteases as early as day 12 in the secondary MC culture (Fig. 5A, 5B). To our surprise, the double-positive cells rapidly increased to 98%–99% at day 18 and 100% at day 21, along with robust cell proliferation. By adopting the previous criteria to determine the intensity of staining [4], we found that ES-derived MCs showed strongly positive staining for chymase from early in the culture period (Fig. 5B, 5D), whereas they stained only weakly for tryptase (Fig. 5A, 5C). At all time points of the secondary MC culture, the percentage of tryptase strongly positive cells did not exceed that of chymase strongly positive ones. These observations were quite different from in vitro-derived human MCs from BM, CB, or fetal liver, in which tryptase was always expressed earlier and more strongly than chymase [19, 21]. Because the mAbs we used to detect tryptase and chymase in monkey ES-derived MCs were specific to human antigens, we confirmed that both mAbs reacted with MCs in cynomolgus monkey skin (Fig. 5E, 5F). These data indicate that the weak reaction against tryptase in monkey ES-derived MCs is not due to low specific binding by the anti-human tryptase mAb.

View larger version (43K): [in this window] [in a new window]   Figure 5. Differentiation of tryptase/chymase double-positive mast cells (MCs) from primate embryonic stem (ES)-derived cobblestone-like cells. (A): A time-course of ES-derived MC differentiation defined by staining with MC-specific tryptase-positive cells. Most of the tryptase-positive cells reacted weakly with the monoclonal antibody (mAb) that recognizes human tryptase. (B): The same time-course analysis as in (A), showing a tendency for time dependently strong expression of chymase in ES-derived MCs. (C): Day 40 ES-derived MCs positively stained with an mAb against human tryptase; more than half of the cells showed weak to mild staining (tryptase strongly positive, 36%). (D): The same day 40 ES-derived MCs as in (C) stained strongly with an mAb to human chymase (chymase strongly positive, 83%). (E): Adult cynomolgus monkey skin section stained with the same tryptase mAb as in (C), showing a strong positive reaction (arrows). (F): The same monkey skin sample stained with chymase mAb, showing an intensity similar to that of tryptase staining (arrows). (G): Reverse transcription-polymerase chain reaction analysis of tryptase and chymase gene expression on ES-derived MCs (day 21), human CB-derived MCs (more than 8 weeks), and a human MC line, HMC-1. Stronger expression of chymase relative to tryptase was observed in ES-derived MCs. (H): A time-course RT-PCR analysis of expression of tryptase and chymase in ES-derived MCs. Chymase mRNA expression appeared earlier (day 4) than tryptase expression (day 8) in the secondary MC culture. Abbreviations: AGM, aorta-gonad-mesonephros; C, chymase; CB, cord blood; ES, undifferentiated embryonic stem cells; G, glyceraldehyde-3-phosphate dehydrogenase; H, hair; NO, no transcripts; T, tryptase.

The Primate ES-Derived MCs Are Functionally Mature CT-MCs
Analysis of intracellular histamine content revealed that ES-derived MCs contained histamine at 7.4 ± 0.5 to 7.6 ± 0.6 pg/cell at day 40 and 10.1 ± 1.8 to 11.3 ± 1.6 pg/cell at day 50. Moreover, in two separate experiments, ES-derived day 50 MCs had a higher histamine content than human CB CD34+ cell-derived day 70 MCs (8.6 ± 1.2 to 9.4 ± 1.1 pg/cell), indicating full maturation of ES-derived MCs (Table 1).

View larger version (40K): [in this window] [in a new window]   Figure 6. Primate embryonic stem (ES)-derived mast cells (MCs) functionally responded to IgE and substance P stimulation. (A): Distribution and negative control of primate ES-derived MCs by fluorescence-activated cell sorting analysis. The upper panel shows SSC and FSC distribution of day 40 cultured primate ES-derived MCs. The lower panel shows the isotope (allophycocyanin) negative staining. (B): When stimulated with 4 µg/ml IgE for 3 days, ES-derived MCs (day 40) upregulated expression of the FccRI by up to 95.8%. (C): Net% β-Hex release was measured after stimulation of the FccRI with mAb CRA-1 or exposure to 10 µM or 100 µM substance P. Human CB-derived MCs (white boxes) showed degranulation after CRA-1 treatment but not after the exposure to substance P, whereas ES-derived MCs (black boxes) responded to substance P in a dose-dependent manner. (D): Net% histamine release. To amplify the response, ES-derived MCs were pretreated with 1 µg/ml IgE overnight before stimulation with CRA-1. ES-derived MCs displayed a dose-dependent histamine release in response to CRA-1, as well as to 10 µM substance P. Abbreviations: APC, allophycocyanin; FSC, forward scatter; β-Hex, β-hexosaminidase; SP, substance P; SSC, side scatter.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

In fetal stages, murine MC precursors are highly concentrated in the yolk sac and fetal blood, suggesting there exists a strong early embryonic wave of MC development [8, 9]. However, because of the impossibility of conducting experimental manipulations on human embryos, little is known about human MC development during the embryonic and fetal stages. Full maturation of CT-MCs is thought to be achieved only after MC progenitors have migrated into the peripheral tissues. Although human CB-derived MCs give rise to tryptase/chymase double-positive MCs in long-term cultures, generally more than 10 weeks in vitro, they still cannot fully mimic the functions of CT-MCs in human skin, such as undergoing degranulation upon stimulation by substance P. Thus, the rapid maturation of primate ES-derived MCs in our present culture system suggests a unique embryonic pathway in primates for the early development of CT-MCs, which may be independent of M-MC development. We simultaneously cultured human CB CD34+ cells under the same culture conditions as in secondary MC cultures of ES-derived CS-like cells with an AGM-S1 feeder layer and found that they developed into MCs through a pathway in which they express tryptase (within 2 weeks) before they express chymase (more than 6 weeks) (data not shown). These data indicate that the primate ES-derived CT-MCs do not follow the same developmental pathway in vitro that has been described for MCs from human hematopoietic progenitors, as shown in MCs derived from CB or BM [21–22] in which tryptase is always expressed earlier than chymase. Rather, the development and maturation of MCs from ES-derived CS-like cells, a highly concentrated fraction of hematopoietic and hemangioblastic progenitors, may represent a unique route that is intrinsically controlled during the early embryonic and fetal stages. Because CT-MCs such as those located in human skin possess a longer life span and higher potential for cell division than do M-MCs, CT-MC precursors may migrate to skin or other peripheral tissues at an early stage during fetal development.

A combination of cytokines, which included SCF, IL-6, and FL, was required to induce the optimal MC development from primate ES cells (Fig. 4C). This cytokine requirement was consistent with human MC development in vitro from CB. Among the cytokines we examined, FL seemed to expand the early hematopoietic pool but did not directly affect MC proliferation, because neither FL alone nor FL in combination with IL-6 could support MC growth, whereas FL+SCF stimulated formation of a few macrophages. FL was only effective in stimulating MC growth in combination with SCF+IL-6. On the other hand, stromal cell support of MC development has been well documented [23–25]. We found that proliferation and differentiation of ES-derived MCs decreased after 3 weeks in primary coculture if the AGM-S1 feeder layer became inadequate (Fig. 4A). ES-derived CS-like cells gave rise to only a few MCs when replated in culture dishes without AGM-S1 cells or on other stromal cells (data not shown). Notably, when day 40 ES-derived mature MCs (in secondary MC culture) were recultured either with AGM-S1 or without a stromal cell layer, the former cultures retained continuous growth while maintaining a chymase-dominant immunochemical phenotype, but the latter cultures gradually decreased in cell numbers and both tryptase and chymase expression was reduced (Fig. 4B; data not shown). These data suggest that AGM-S1 cells support both proliferation and maturation of the primate ES-derived CT-MCs, although the development of MCs from primate ES cells is not necessarily AGM-S1 cell-dependent.

Through our experiments, we could not confirm the specific progenitor type from which the ES-derived MCs arose. Nor could we determine what phenotype a committed ES-derived MC precursor possessed. Since a committed hematopoietic stem/progenitor cell has not yet been found in murine, human, or nonhuman primate ES cells, and because of the limited antibodies and genetic information specific for monkey ES cells and their progeny, pinpointing an ES-derived MC precursor has proven difficult. Although murine ES-derived functional MCs have been reported [26], because of considerable heterogeneity between rodent and human MCs, understanding of early human MC development using human and nonhuman primate ES cells is important. In this newly established in vitro culture system, large quantities of mature CT-MCs can be produced from primate ES cells in a relatively short time. Thus, our culture system will provide an excellent tool to further investigate the mechanisms that control early development of MCs in both human and nonhuman primates.

	Summary

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

 
We have illustrated a novel culture system for efficient production of CT-MCs with phenotypic and functional maturity from primate ES cells. The rapid maturation of ES-derived MCs suggests a unique embryonic pathway in primates for early development of CT-MCs, which may be independent from the developmental pathway of M-MCs. Our discovery should facilitate the further exploration of the unknown mechanisms that control early development of MCs in both human and nonhuman primates.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

 
We thank Dr. Y. Horiguchi (Osaka Redcross Hospital, Osaka, Japan) for conducting the electron microscopic observations. We also thank Prof. T. Nakano at Osaka University and Profs. H. Nakauchi, T. Kitamura, and S. Watanabe at the Institute of Medical Science, University of Tokyo, for constructive discussions. This work was supported in part by the Establishment of International COE for Integration of Transplantation Therapy and Regenerative Medicine and by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. F.M. and N.K. contributed equally to this work.

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