Bone Morphogenetic Protein-Mediated Modulation of Lineage Diversification During - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:56 |只看该作者 |倒序浏览 |打印

作者：Gudrun Gossraua,b, Janine Thieleb, Rachel Konanga, Tanja Schmandta, Oliver Brstlea作者单位：aInstitute of Reconstructive Neurobiology, Life  Brain Center, University of Bonn and Hertie Foundation, Bonn, Germany;bDepartment of Neurology, University of Dresden Medical Center, Dresden, Germany




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      【摘要】3 ]7 m4 d/ Y; z. o! x/ X. S8 Z
   Embryonic stem cells (ES cells) can give rise to a broad spectrum of neural cell types. The biomedical application of ES cells will require detailed knowledge on the role of individual factors modulating fate specification during in vitro differentiation. Bone morphogenetic proteins (BMPs) are known to exert a multitude of diverse differentiation effects during embryonic development. Here, we show that exposure to BMP2 at distinct stages of neural ES cell differentiation can be used to promote specific cell lineages. During early ES cell differentiation, BMP2-mediated inhibition of neuroectodermal differentiation is associated with an increase in mesoderm and smooth muscle differentiation. In fibroblast growth factor 2-expanded ES cell-derived neural precursors, BMP2 supports the generation of neural crest phenotypes, and, within the neuronal lineage, promotes distinct subtypes of peripheral neurons, including cholinergic and autonomic phenotypes. BMP2 also exerts a density-dependent promotion of astrocyte differentiation at the expense of oligodendrocyte formation. Experiments involving inhibition of the serine threonine kinase FRAP support the notion that these effects are mediated via the JAK/STAT pathway. The preservation of diverse developmental BMP2 effects in differentiating ES cell cultures provides interesting prospects for the enrichment of distinct neural phenotypes in vitro." `8 }. J# \7 K& V

Disclosure of potential conflicts of interest is found at the end of this article. . [2 F3 m: Z8 H, _; Y7 T! d/ U
      【关键词】 Embryonic stem cells Neural crest Bone morphogenetic protein Peripheral neurons7 I* C- v- ]8 j2 Z
   INTRODUCTION9 z$ e7 V. l$ W3 ~6 f* F2 A8 K# \8 U0 A) s

In vitro differentiation of embryonic stem (ES) cells into tissue-specific precursors provides a powerful tool for the study of lineage segregation and the generation of donor cells for transplantation. Upon aggregation, ES cells differentiate into EBs, which give rise to cell types of all three germ layers. Although to date the regulation of germ layer specification in ES cell cultures remains poorly understood, several studies indicate that the hierarchy of somatic lineage decisions observed during EB differentiation replicates many aspects of in vivo embryonic development .

We and others have developed growth factor-based protocols that permit the gradual differentiation of EBs into different neural precursor cell populations .$ H. v7 M& `! e% n% b
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Bone morphogenetic proteins (BMPs) play a key role in normal development and in vitro stem cell differentiation .
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In this study, we set out to explore whether and to what extent treatment of EBs, FGF2-treated early pan-neural precursors, and FGF2/EGF-expanded late neural precursors with BMP2 promotes the generation of peripheral neural cells and other phenotypes. Our data show that exposure of EBs to BMP2 inhibits neuronal differentiation and promotes the generation of non-neural ectoderm, as well as mesoderm and neural crest cell fates. In EB-derived early neural precursors, BMP2 induces distinct peripheral nervous system (PNS) fates and promotes astrocytic lineage differentiation. BMP2 treatment of FGF2/EGF-propagated late neural precursors revealed that this promotion of astrocyte differentiation occurs at the expense of oligodendrocyte development. The preservation of these developmental effects and their underlying mechanisms in different stages of neural ES cell differentiation provides interesting prospects for enhancing the derivation of biomedically relevant cell types in vitro.  y9 Y" {! T0 e1 u8 v
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MATERIALS AND METHODS

ES Cell Differentiation

In vitro differentiation of mouse ES cells (line J1 ; differentiation was induced by growth factor withdrawal (ESNPFGF2-EGF-DIFF).8 ?6 M4 |9 ^' S% v  _5 g$ d
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BMP and Rapamycin Treatment
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Cells were exposed to BMP2 (R&D Systems) at different stages of in vitro differentiation (details are given in flow charts in Figs. 1¨C4). Initial dose-finding experiments revealed that exposure of EBs or ESNPFGF2-EGF to 10 ng/ml BMP2 impaired generation and viability of the neural precursors, respectively. Thus, experiments at these differentiation stages were performed with 5 ng/ml BMP2. EBs were subjected to RNA extraction either directly after a 3-day-exposure to BMP2 or after additional plating in ITS medium and subsequent differentiation (Fig. 1, ESNPITS-DIFF). In some experiments, BMP2-treated EBs were further propagated in growth factor-containing media as described above. For analysis of BMP2 effects on FGF2-propagated neural precursors, ESNPs proliferated for 4 days in FGF2 were cultured in the presence of FGF2 and 10 ng/ml BMP2 for another 4 days. BMP2 treatment of ESNPFGF2-EGF-PROL cultures was done for 2 days at a concentration of 5 ng/ml. Density-dependent induction of astrocytes and smooth muscle cells was studied at plating densities of 105 cells per cm2 and 103 cells per cm2. Rapamycin (Sigma-Aldrich) was applied at a concentration of 1 µM. For Western blot and immunofluorescence analysis of STAT and Smad phosphorylation, cells were withdrawn from FGF2 in the presence of BMP2.  U- F( R/ I/ W$ {

Figure 1. Effects of BMP2 on early embryonic stem (ES) cell differentiation. (A): Schematic illustration of the ES cell differentiation protocols. ES cells were aggregated to EBs and subjected to a stepwise differentiation protocol involving ITS medium and sequential proliferation of ESNPs in FGF2 and FGF2/EGF. Differentiation was induced by 4-day growth factor withdrawal (DIFF). (B): Reverse transcription-polymerase chain reaction (RT-PCR) analysis of BMP receptor transcripts relevant for neural differentiation ( , positive control: E16 mouse embryo; ¨C, no template control). (C): BMP2 treatment of EBs resulted in an induction of brachyury (mesoderm) and FGF5 (ectoderm) and a downregulation of the neuronal marker ßIII-tubulin in quantitative RT-PCR analysis. (D): Upon plating and differentiation in ITS medium (EBITS-DIFF in ), BMP2-treated EBs showed enhanced expression of neural crest markers. Abbreviations: AFP, -fetoprotein; BMP, bone morphogenetic protein; BMPR, bone morphogenetic protein receptor; DIFF, differentiated; EGF, epidermal growth factor; ESNP, embryonic stem cell-derived neural precursor; FGF, fibroblast growth factor; PROL, proliferated; SMA, smooth muscle actin.
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5-Bromo-2'-deoxyuridine Incorporation Assay8 }8 f# e2 L1 T% r% F
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Concomitantly with the onset of BMP2 exposure, ESNPFGF2-PROL cultures were treated with 10 µM 5-bromo-2'-deoxyuridine (BrdU) (Sigma-Aldrich) for a total period of 4 days. After fixation, cells were processed for double immunofluorescence using primary antibodies to BrdU (BD Biosciences, Heidelberg, Germany, http://www.bdbiosciences.com; 1:100), glial fibrillary acidic protein (GFAP) (1:200; Dako, Hamburg, Germany, http://www.dako.com), and smooth muscle actin (SMA) (1:200; Dako).
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Immunofluorescence Analysis

The cultures were fixed with 4% parafomaldehyde in phosphate-buffered saline (PBS) for 10 minutes and incubated with primary antibodies to nestin (Rat-401; 1:200; Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/dshbwww), ßIII-tubulin (TUJ1; 1:500; Covance, Berkley, CA, http://www.covance.com), p75 (1:1,000; Chemicon), peripherin (1:200; Chemicon), Mash1 (1:200; RDI, Concord, MA, http://www.researchd.com), Brn3.0 (1:200; Chemicon), Sox10 (1:3; a gift from L. Sommer), Ret (1:10; R&D Systems), O4 (1:200; R&D Systems), P0 (1:50; a gift from A. Baron-van Evercooren), SMA (1:200; Dako), GFAP (1:200; Dako), choline acetyltransferase (ChAT; 1:200; Chemicon), calretinin (1:200; Biozol), Islet-1 (isl-1; 1:50; Developmental Studies Hybridoma Bank), serotonin (5HT; 1:1,000; AbD Serotec, Oxford, U.K., http://www.ab-direct.com), GABA (1:200; Sigma-Aldrich), phosphorylated Smad1 (1:200; Upstate, Charlottesville, VA, http://www.upstate.com), or tyrosine hydroxylase (TH; 1:200; Sigma-Aldrich) for 2 hours in PBS containing 1% normal goat serum. Triton X-100 (0.1%; Sigma-Aldrich) was added for labeling intracellular antigens. Detection of primary antibodies was performed with appropriate fluorescein isothiocyanate-, Cy3-, or rhodamine-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com). Microscopical analysis was performed using Zeiss Axioskop 2 or Zeiss LSM510 microscopes.) c/ Y# k9 G; }

Western Blot

Nuclear extracts were prepared with NE-PER nuclear and cytosolic extraction reagents (Pierce, Rockford, IL, http://www.piercenet.com) according to the supplier's instructions using protease and phosphatase inhibitors. Normalized protein lysates including positive controls were loaded on 10% polyacrylamide gels and blotted on nitrocellulose membranes. Blots were probed with antibodies directed against phosphorylated STAT3 (Ser 727; Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com), phosphorylated Smad1 (Cell Signaling Technology), and appropriate alkaline phosphatase-conjugated secondary antibodies (Sigma-Aldrich). Signal was detected by chemiluminescence.2 R' ]! h! v0 K3 M5 \

Immunoprecipitation

Total cell lysates were produced as described elsewhere . Normalized lysates were incubated with antibodies against FKBP12 or FRAP overnight at 4¡ãC. After incubation with protein G-Sepharose beads (GE Healthcare, Little Chalfont, Buckinghamshire, U.K., http://www.gehealthcare.com/), immune complexes were washed, heated at 70¡ãC, separated by SDS-polyacrylamide gel electrophoresis, and transferred onto nitrocellulose. Blots were probed with antibodies against FKBP12 (Transduction Laboratories, Lexington, KY, http://www.bdbiosciences.com/pharmingen), BMP receptor 1A (BMPR 1A; R&D Systems), FRAP (Transduction Laboratories), and STAT3 (Cell Signaling Technology).5 h1 E6 v' v: c, u3 K  s- V5 j

Quantification and Statistical Analysis
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Quantification of labeled cells was based on 20 high-power fields in each of three independent experiments, with a minimum of 1,000 cells counted per experiment. Data are presented as mean values ¡À SEM. Statistical analyses were done using the 2 test; p values 0.05 were regarded significant. In the case of low filled cells, a two-tailed Fisher's exact test was performed.& ]' s1 y. g" F7 w, b) t' P
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Reverse Transcription-Polymerase Chain Reaction and Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction Analysis$ L9 x9 q& a3 U1 @5 V. Q8 f
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For reverse transcription-polymerase chain reaction (RT-PCR) analysis, cultures were harvested in Trizol (Invitrogen) for isolation of RNA, which was then used for first-strand cDNA synthesis (Superscript II; Invitrogen). RT-PCR was performed using primers for ß-actin , ßIII-tubulin (s, CAGATGCTGCTTGTCTTGGC; as, GATGATGACGAGGAATCGGAA), GFAP (s, GAGTACCACGATCTACTCAAC; as, CCACAGTCTTTACCACGATGT), Sox10 (s, AGG TCA AGA AGG AAC AGC AG; as, TAC TGG TCG GCT AGC TTT CT), BMPR IA (s, GCTCCATGGCACTGGTATG; as, GCAATAGTTCGCTGAACC), BMPR IB (s, GCCTGCCATAAGTGAGAAGC; as, ACAGGCAACCCAGAGTCATC), BMPR II (s, CTCTGAGCATTCGATGTCCAG; as, CAAGCTAGAACTGGTACTGC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (s, ACGACCCCTTCATTGACCTCAACT; as, ATATTTCTCGTGGTTCACACCCAT), BF-1 (s, ATGACTTCGCAGACCAGCAC; as, AACGTTCACTTACAGTCTGG), brachyury (s, CCGGTGCTGAAGGTAAATGT; as, CCTCCATTGAGCTTGTTGGT), FGF5 (s, GAAAAGACAGGCCGAGAGTG; as, GAAGTGGGTGGAGACGTGTT), AFP (s, AAAATTTGGATCCCGAAACC; as, TGCGTGAATTATGCAGAAGC), Mitf (s, AACCGACAGAAGAAGCTGGA; as, TGATGATCCGATTCACCAGA), Pax6 (s, AACAACCTGCCTATGCAACC; as, CTTGGACGGGAACTGACACT), and Hb9 (s, ACAGGCGGCTCTCTATGGGACA; as, TTCCCCAAG AGGTTCGACTGC).! J" g" o! F* w1 h- d/ W* R1 }( r

All RNA probes underwent a DNase digestion (Promega, Mannheim, Germany, http://www.promega.com). In cases of non-exon-spanning primer pairs, a PCR without the reverse transcription step was performed to exclude contaminating genomic DNA. Quantitative real-time RT-PCR was carried out using the iCycler System (Bio-Rad, Munich, Germany, http://www.bio-rad.com), and amplification was monitored and analyzed by measuring the binding of the fluorescent dye SYBR Green I to double-stranded DNA. Expression of GAPDH was used for normalization.

RESULTS

Differential Expression of BMP Receptors During Neural ES Cell Differentiation% m1 C2 x2 F; M: `' }

ES cells were aggregated to EBs to induce differentiation and subsequently propagated in ITS medium, which favors the survival of neuroepithelial cells (Fig. 1A). To enrich for neural precursors, cells were first propagated in the presence of FGF2 (ESNPFGF2-PROL) .1 V$ l5 Q3 J5 X% s
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Promotion of Neural Crest Generation in BMP-Treated EBs# ~: d( j2 i( J  d/ T, n

Following up on the early expression of BMP receptors in the EB stage, we first studied whether stimulation of EBs with BMP2 affects the expression of germ layer markers. A 3-day exposure of EBs to BMP2 led to a strong increase of the mesoderm marker brachyury (Fig. 1C). EBs showed spontaneous expression of ßIII-tubulin, indicating differentiation of neuroectodermal cells. BMP2 increased the ectoderm marker FGF5, whereas it decreased the expression of the neuronal differentiation marker ßIII-tubulin (Fig. 1C). Similar results were obtained using a different ES cell line (CJ7; data not shown). EBs also showed expression of Sox10, an early neural crest marker. This provides the possibility that neural crest cells (NCCs) may exist in EBs, and therefore the treatment of EBs with BMP2 might increase putative NCCs. Therefore, we tested whether BMP2 treatment at the EB stage affects the elaboration of neural crest fates in subsequent culture steps. Upon plating and culture in ITS neural selection medium (Fig. 1A, EBITS-DIFF), BMP-treated EBs gave rise to cells with enhanced expression of several neural crest-related genes, including Snail, Sox10, p75, Mitf, and SMA (Fig. 1D). Snail is expressed in both premigratory and¡ªtogether with Sox10 and p75¡ªmigrating neural crest precursors . Considering the strong induction of the mesoderm marker brachyury in BMP-treated EBs and the only moderate increase of p75 transcripts in the EBITS-DIFF cultures, it appears likely that both routes contribute to the strong induction of SMA.

Inhibition of Neuronal Differentiation Coincides with an Upregulation of PNS Markers& L- M$ _  U  {

The pronounced inhibition of neuronal differentiation in BMP-treated EBs was not restricted to the EB stage but was preserved in all subsequent culture steps. For instance, ESNPFGF2-DIFF cultures derived from BMP2-treated EBs contained only 21% ¡À 2% ßIII-tubulin-positive neurons compared with 63% ¡À 4% in ESNPFGF2-DIFF cultures derived from untreated EBs (supplemental online Fig. 6). Inhibition of neuronal differentiation was also observed when BMP2 was added to early ES cell-derived neural precursors proliferating in FGF2 (Fig. 2A, 2B). Following growth factor withdrawal, only 28% ¡À 0.6% of BMP2-treated ESNPFGF2-DIFF gave rise to ßIII-tubulin-positive neurons, compared with 49% ¡À 1.5% of their untreated counterparts (Fig. 2B). A pronounced decrease in neuronal differentiation was also noted upon treatment with 10 ng/ml BMP4 (data not shown). On the other hand, within the decreased fraction of neurons originating from BMP-treated ESNPFGF2-DIFF, the percentage of peripherin-positive cells increased from 45.6% ¡À 3% to 66.6% ¡À 2% (Fig. 2C, 2D). Such an increase in peripherin-positive cells could also be observed in ESNPs derived from a different ES cell line (CJ7; data not shown). Both the overall decrease in neuronal differentiation and the relative increase of peripherin-positive neurons were abolished by cotreatment with noggin, a known BMP inhibitor (Fig. 2B, 2C). Since we were interested in the generation of peripheral neurons, we investigated the differentiated cells for coexpression of peripherin and ßIII-tubulin. Dose-response experiments indicated that BMP2-mediated promotion of peripherin- and ßIII-tubulin-positive cells saturated at 10 ng/ml BMP2 (Fig. 2D). Data from quantitative RT-PCR confirmed the increase of peripherin and the decrease of ßIII-tubulin transcripts following BMP2 treatment (Fig. 2E, 2F). Furthermore, the expression of BF-1, a transcription factor typically expressed in the telencephalon, and Pax6, a neural progenitor marker within telencephalon and intermedioventral spinal cord, was downregulated in BMP2-treated ESNPFGF2-DIFF (Fig. 2E). In contrast, c-ret and Mash1, which are expressed in a subset of peripheral neurons, were upregulated upon BMP2 treatment. We also observed that BMP2 induced an increased expression of the neural crest marker snail.: Q" `, G& J, ~* o% j6 W0 r- c; o: [

Figure 2. BMP2 inhibits neuronal differentiation and promotes peripherin-positive fates in ESNPs. BMP2 treatment during FGF2-mediated proliferation (A) decreased overall neuronal differentiation in ESNPFGF2-DIFF cultures (B). At the same time, the ratio of peripherin-expressing neurons increased markedly (C). Both effects were abolished by noggin treatment. (D): Dose-dependent effect of BMP2 on the generation of peripherin-expressing neurons. (E): Quantitative reverse transcription-polymerase chain reaction analyses confirmed a decrease in ßIII-tubulin transcripts and showed a concomitant induction of the neural crest-associated markers c-ret, snail, and Mash1. At the same time, Pax6 and BF-1 were downregulated. (F): Representative anti-peripherin/ßIII-tubulin double immunofluorescence of a BMP-treated ESNPFGF2-DIFF culture. Scale bar = 20 µm. Abbreviations: BMP, bone morphogenetic protein; DIFF, differentiated; ESNP, embryonic stem cell-derived neural precursor; FGF, fibroblast growth factor.+ S* j# [. A! A$ n$ g

BMP2 Modulates the Subspecification of Peripherin-Positive ES Cell-Derived Neurons, d) I& g( R* [7 ]. s7 N. u( u
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We next studied to what extent BMP2 exposure during FGF2-mediated proliferation influences the segregation of peripherin-expressing neurons in ESNPFGF2-DIFF cultures. We found that BMP2 treatment increased ChAT-positive cells within the peripherin-positive fraction (12.4% ¡À 2% vs. 6.7% ¡À 2%; Fig. 3B, 3C, 3J). At the same time, exposure to BMP2 decreased the number of peripherin/Islet-1-positive neurons. Islet-1 (isl-1) is a transcription factor that is enriched not only in cholinergic spinal cord motoneurons but also in a subgroup of dorsal root ganglion cells. Thus, our findings raised the possibility that the increase of ChAT-positive cells induced by BMP2 reflected the promotion of cholinergic PNS neurons rather than an increase of spinal cord motoneurons. To test this possibility, we investigated the expression of the homeobox gene Hb9, a specific marker of motoneurons . We further noticed an increase of peripherin-positive neurons expressing GABA (6.1% ¡À 1% vs. 2.2% ¡À 1%; Fig. 3B, 3G) and, to a lesser extent, serotonin (1.2% ¡À 0.2% vs. 0.2% ¡À 0.2%; Fig. 3B, F). A subset of the serotonin/peripherin-positive neurons was also found to express ChAT (data not shown). Within the peripherin-positive population, BMP2 further favored the development of putative Mash1-positive autonomic neurons (2.7% ¡À 1.2% vs. 0.7% ¡À 0.5%; Fig. 3B, 3H). In contrast, BMP2 treatment resulted in slightly but insignificantly decreased numbers of TH/peripherin-positive putative catecholaminergic PNS neurons and Brn3.0/peripherin-positive presumptive sensory phenotypes (Fig. 3B, 3E). Quantitative RT-PCR confirmed the increase of ChAT and the decrease of isl-1 expression upon BMP2 treatment (Fig. 3C). Thus, BMP2 has diverse effects on the subspecification of ES cell-derived peripherin-positive neuronal progeny.! q6 h; J+ z8 j6 f+ Z1 P
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Figure 3. BMP2 modulates the fate of peripherin-positive embryonic stem cell-derived neurons. (A, B): BMP2 treatment during FGF2-mediated proliferation of ESNPs increased the number of peripherin/ChAT-expressing cells in their differentiated progeny. At the same time, BMP2 decreased the peripherin/isl-1-positive neurons. BMP2 exposure further favored the generation of peripherin-positive neurons co-expressing calretinin, GABA, 5HT, and Mash-1. No significant differences in the emergence of peripherin/TH-expressing and peripherin/Brn3.0-positive neurons were observed between the BMP2-treated and control group. (C): Quantitative reverse transcription-polymerase chain reaction analysis confirmed the BMP effects on ChAT, Hb9, and isl-1 expression. (D¨CJ): Representative examples of neurons co-expressing ChAT and isl-1 (D), peripherin and Brn3.0 (E), serotonin (F), GABA (G), Mash1 (H), calretinin (I), and ChAT (J). Scale bars = 10 µm (D, E, G¨CJ) and 20 µm (F). Single channel captures are given in supplemental online Figure 7. Abbreviations: 5HT, serotonin; BMP, bone morphogenetic protein; ChAT, choline acetyltransferase; DIFF, differentiated; FGF, fibroblast growth factor; GABA, -aminobutyric acid; PNS, peripheral nervous system; TH, tyrosine hydroxylase., Z: M6 g# p  y2 x. Q/ c: s; F

The BMP2 Effect on Astrocyte and Smooth Muscle Differentiation Is Density-Dependent
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The results of previous studies indicate that BMP signaling promotes the development of GFAP-positive glia and smooth muscle phenotypes from primary neuroepithelial precursors  argue for an instructive role of BMPs on astrocyte and smooth muscle differentiation, the changes we observed could also be due to a selective mechanism against other cell fates. To distinguish between these two possibilities, cells were subjected to a proliferation assay. In high-density cultures, BMP2 caused a decrease in BrdU-labeled cells from 50% ¡À 2.3% to 27% ¡À 2.5% (supplemental online Fig. 8). Similarly, BMP2-treated low-density cultures showed a reduction of BrdU-positive cells (66% ¡À 2.3% vs. 45% ¡À 2.5%; supplemental online Fig. 8). No increase of BrdU/GFAP and BrdU/SMA double-labeled cells was observed in high- and low-density cultures, respectively. These findings argue against a selective promotion of astrocyte or smooth muscle proliferation by BMP2. Furthermore, an increase in astrocyte differentiation was also observed when BMP2 was solely added during FGF2 withdrawal (data not shown), indicating that this effect does not depend on proliferation imposed by exogenous FGF2 treatment.) i; e5 b0 R# z. C

Figure 4. Density-dependent promotion of astrocyte and smooth muscle differentiation in BMP2-treated ESNPs. HD and LD cultures of FGF2-propagated ESNPs were exposed to 10 ng/ml BMP2 and subsequently differentiated by growth factor withdrawal (A1 A2). In HD cultures (plating density, 105 cells per cm2), BMP2 resulted in a clear increase of astrocyte differentiation (B, C). In contrast, LD cultures (plating density, 103 cells per cm2) responded with increased smooth muscle differentiation (D, E). These findings were confirmed by qRT-PCR analysis of GFAP and SMA expression (F). Simultaneous application of noggin and BMP2 abolished both effects (B, E). Rapamycin inhibited BMP2-mediated astrocyte induction in HD cultures, whereas smooth muscle differentiation in LD cultures remained unaffected (B, E). (G¨CI): BMP treatment also promoted astrocyte differentiation in ESNPFGF2-EGF-PROL cultures. This effect occurred at the expense of oligodendrocyte formation. Co-application of BMP2 and rapamycin reduced astrocyte differentiation to subcontrol levels and restored oligodendrocyte differentiation as shown by quantitative (G) and qualitative (I) assessment of GFAP and O4 expression. Scale bars = 30 µm (C), 25 µm (D), and 10 µm (I). Abbreviations: BMP, bone morphogenetic protein; DIFF, differentiated; EGF, epidermal growth factor; FGF, fibroblast growth factor; GFAP, glial fibrillary acidic protein; HD, high-density; LD, low-density; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; Rapa, rapamycin; SMA, smooth muscle actin.
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We wondered whether the effect of BMP2 on astrocyte differentiation is maintained at later stages of neural differentiation. In our experimental set-up, EB-derived early neural precursors proliferate as multipotent ESNPFGF2-PROL cultures . Under standard conditions, ESNPFGF2-EGF-DIFF cultures contained 72% ¡À 3% GFAP-positive astrocytes and 14.8% ¡À 2% O4-expressing oligodendrocytes. BMP2 exposure during FGF2/EGF application increased the number of astrocytes (89.4% ¡À 2.4%), while decreasing that of oligodendrocytes (0.6% ¡À 1%; Fig. 4G). A similar increase in GFAP-positive cells at the expense of oligodendrocyte differentiation was detected upon treatment with BMP4 or in ESNPFGF2-EGF-DIFF cultures prepared from a different ES cell line (CJ7; data not shown). ESNPFGF2-EGF-PROL cultures exposed to BMP2 also gave rise to 1.6% ¡À 0.4% SMA-positive smooth muscle cells, which were undetectable in non-BMP-treated cultures. P0-positive putative Schwann cells, ranging from 1% to 3% of the cells in untreated ESNPFGF2-EGF-DIFF cultures, were no longer detectable after BMP2 treatment (not shown).6 b! ?2 I/ ^; `. G5 m( V% U) [: w1 j9 ^5 H
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Differential Activation of the JAK/STAT and Smad Pathways in Astrocyte and Smooth Muscle Specification  B* ~3 {% [8 S: L: P

Recent data on primary neural precursor cells suggest that BMP4 activates distinct signaling pathways in a density-dependent manner . Consistent with this scenario, co-immunoprecipitation of BMPR IA and FKBP12 from ESNPFGF2-DIFF was reduced following BMP2 treatment (Fig. 5J). Concomitantly, co-immunoprecipitation of STAT3 and FRAP was increased (Fig. 5K, 5L)." o$ ~6 V2 o; \# x" [$ m/ ^! a

Figure 5. Cross-signaling between BMPR and the JAK/STAT pathway. Recent evidence from primary cell cultures suggests that BMP2-mediated astrocyte induction in high-density cultures is executed via the serine threonine kinase FRAP and cross-activation of the JAK/STAT pathway (). Cotreatment with BMP2 and the FRAP inhibitor rapamycin was used to test for this hypothesis. Indeed, BMP treatment increased the level of STAT3 phosphorylation in high-density cultures, an effect that could be abolished by rapamycin (B) ( , positive control: 2-day-LIF-treated ESNPFGF2-EGF-PROL). Vice versa, BMP2 led to an increase in phosphorylated Smad1 in low-density cultures, giving rise to smooth muscle cells (C). Cross-signaling between BMPR and the JAK/STAT pathway was further supported by a decreased immunoprecipitation of FKBP12 and BMPR IA (D) and an increased immunoprecipitation of FRAP and STAT3 (E, F) (quantification via chemiluminescence) after BMP2 treatment. Both increased STAT3 phosphorylation and co-immunoprecipitation of FRAP and STAT3 cold be abolished by rapamycin (B, E). Abbreviations: BMP, bone morphogenetic protein; BMPR, bone morphogenetic protein receptor; DIFF, differentiated; ESNP, embryonic stem cell-derived neural precursor; FGF, fibroblast growth factor.
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As in the ESNPFGF2 cultures, addition of rapamycin to BMP2-treated ESNPFGF2-EGF cultures abolished the effect of BMP2 on astrocyte differentiation, yielding fewer GFAP-positive cells than in rapamycin-untreated cultures (52.4% ¡À 7% vs. 72% ¡À 3%). At the same time, rapamycin rescued oligodendroglial differentiation (Fig. 4G, 4I), restoring the level of O4-positive cells to 13% ¡À 1.5%. In contrast, rapamycin had no effect on the number of SMA-positive smooth muscle and P0-immunoreactive Schwann cells as compared with non-rapamycin-treated BMP2-exposed ESNPFGF2-EGF-DIFF cultures. Due to poor survival at low density, these experiments could only be performed in high-density cultures, yet the data indicate that BMP2-mediated promotion of astrocyte induction is maintained in ES cell-derived glial precursors and occurs at the expense of oligodendroglial differentiation. The neutralization of the BMP2 effect by rapamycin suggests that astrocyte induction at the glial precursor stage, too, is mediated via cross-signaling between the Smad and JAK/STAT pathway.

DISCUSSION
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The results of this study show that BMP2 exerts a variety of diverse and stage-specific effects on the in vitro differentiation of murine ES cells. The data obtained have implications on several levels. From a biomedical point of view, the effects of BMP2 on ES cell differentiation may provide a basis for the enrichment of specific phenotypes for cell replacement strategies. In particular, BMP2 treatment of differentiating ES cell cultures may be used for the enrichment of therapeutically relevant peripheral neural phenotypes. From a developmental perspective, our data indicate that many of the BMP2-mediated differentiation events observed during embryonic development are preserved in differentiating ES cell cultures, although clonal analyses will be required to fully delineate the differentiation potential of the various BMP-induced precursor cell stages and their lineage relationship. Finally, on a mechanistic level, our data give further support to the hypothesis that some of the BMP2-induced differentiation effects are based on a cross-signaling between the Smad and the JAK/STAT pathways.
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Early Effects: Inhibition of Neuronal Differentiation and Promotion of Neural Crest Fates
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BMP signaling plays a key role in early embryonic patterning. The BMP2-induced differentiation events observed in the early stages of our ES cell differentiation paradigm are in line with these developmental effects. BMP2 application to differentiating ES cells promotes the expression of ectodermal and mesodermal markers while suppressing neuronal differentiation. Similar findings have been made in ES cell differentiation paradigms involving retinoic acid and stromal cell cocultures .
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During neurulation, the ectoderm divides into neural plate and prospective epidermis. The boundary region in-between is the source of the neural crest. The induction of this transient population is influenced by many factors, including Wnt signals, FGF, retinoic acid . In keeping with this, exposure of EBs to BMP2 resulted in a pronounced increase of neural crest marker expression in EB-derived neural cells.* Q& k) r9 Y+ m: M. ]" o9 m" T
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Elaboration of Different Peripheral Neuronal Subtypes

Promotion of neural crest differentiation by BMP2 was not restricted to EBs but could also be elicited in FGF2-propagated ESNPs. Despite the overall decrease of ßIII-tubulin-positive neurons in BMP2-treated ESNPFGF2-DIFF cultures, we noted a relative increase of the fraction of peripherin-positive neurons. In contrast, the generation of peripherin-positive neurons form BMP-treated EBs was highly variable and inefficient. We also found a decreased expression of the telencephalic transcription factor BF-1, suggesting that in our model, BMP2 favors the generation of peripheral neurons at the expense of anterior CNS neurons. Pax6, too was downregulated by BMP2, a phenomenon compatible with the dorsalizing effect of BMPs in the developing neural tube .
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There is evidence that BMPs not only promote the differentiation of peripherin-expressing neurons but also modulate their subspecification. In primary neural crest stem cells, BMPs have been shown to induce cholinergic neurons .
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In primary neural crest cells, BMPs have been shown to inhibit peripheral sensory neuron differentiation , indicating once again that depending on time and dosage, BMPs may have diverse effects on neural differentiation.

Other effects of BMP2 on the subspecification of peripherin-positive neurons include induction of peripherin/Mash1- and peripherin/GABA-neurons.
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Mechanisms of BMP-Mediated Astrocyte Induction2 w7 a6 h- I* ~+ n' U" K

Studies on primary neural precursors indicate that BMPs promote astrocytic differentiation while inhibiting the development of oligodendrocytes . In these cultures, BMP2 also led to a decrease in oligodendroglial phenotypes. Thus, BMP-mediated promotion of astrocyte differentiation and inhibition of oligodendrocyte development can be translated to ES cell-derived neural precursors.
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Remarkably, in FGF2-propagated ES cell-derived neural precursors, the effect of BMP2 on astrocyte differentiation was strictly density-dependent. Whereas BMP2 induced astrocytic differentiation in high-density cultures, it promoted smooth muscle development in low-density cultures. Similar observations have been made by others in BMP-treated primary neural precursors .( L+ Y* a. {# P0 H, a
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Perspectives for Peripheral Nervous System Repair

The results of this study indicate that a variety of BMP-mediated effects on germ layer differentiation, neural crest induction, and neural sublineage segregation observed during normal development can be recapitulated during the stepwise differentiation of ES cells into neural precursors (supplemental online Fig. 9). So far, BMP-related studies on ES cells have mainly focused on maintenance of pluripotency . In that regard, BMP-mediated generation of ES cell-derived peripheral neurons could serve as a starting point for the generation of disease-specific donor cells.+ Z- ^5 R  e0 \* h  ~/ d

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST% ?5 B5 f; ]* j; O$ e

The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS
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This work was supported by the DFG (Go-1066/1-1), the Hertie Foundation, BONFOR, and European Union project LSHB-CT-2003-503005 (EuroStemCell). We gratefully acknowledge Heinz Reichmann, Andrea Kempe, Anke Altkr¨¹ger, Hassan Mziaut, Cemile Jakopoglu, Sabine Schenk, and Barbara Steinfarz for advice and technical support and L. Sommer for helpful discussions.4 d5 J) L# k8 L0 J4 n1 v% ^
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