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Cell Biology International (2012) 36, 267–271 (Printed in Great Britain)
Wnt3a is involved in the early stage of miPSC and mESC haemopoietic differentiation
Wencheng Zhang, Hailei Yao, Sihan Wang, Shuangshuang Shi, Yang Lv, Lijuan He, Xue Nan, Wen Yue, Yanhua Li1 and Xuetao Pei1
Stem Cell and Regenerative Medicine Lab, Beijing Institute of Transfusion Medicine, Beijing 100850, Peoples Republic of China


The Wnt/β-catenin signalling pathway is important in regulating not only self-renewal of haemopoietic progenitors and stem cells but also haemopoietic differentiation of ESCs (embryonic stem cells). However, it is still not clear how it affects haemopoietic differentiation. We have used a co-culture system for haemopoietic differentiation of mouse ESCs and iPSCs (induced pluripotent stem cells) in which the Wnt3a gene-modified OP9 cell line is used as stromal cells. The number of both Flk1+ and CD41+ cells generated from both co-cultured mouse ESCs and mouse iPSCs increased significantly, which suggest that Wnt3a is involved in the early stages of haemopoietic differentiation of mouse ESCs and mouse iPSCs in vitro.


Key words: CD41, differentiation, mouse induced pluripotent stem cell (iPSC), OP9 cell, Wnt3a

Abbreviations: EB, embryoid body, ESC, embryonic stem cell, FBS, fetal bovine serum, GFP, green fluorescent protein, iPSC, induced pluripotent stem cell, mESC, mouse ESC, miPSC, mouse iPSC, PE, phycoerythrin, RT–PCR, reverse transcription–PCR

1Correspondence may be addressed to either of these authors (email shirlylyh@126.com or peixt@nic.bmi.ac.cn).


1. Introduction

For decades, ESCs (embryonic stem cells) have been used as a system for early development studies. Hopes have been raised for ESCs to become a universal resource for cellular replacement therapies in the clinical treatment of diseases (Keller, 2005). ESCs, derived from the inner cell mass of mammalian blastocysts, can extensively proliferate, maintain an undifferentiated state and differentiate into all kinds of cells derived from the three germ layers, including haemopoietic lineage in vitro. However, the difficulty of generating patient- or disease-specific ESCs raises the problem of immune rejection when ESCs are used in clinical transplantation. Ethical controversies also hinder the progress of their practical application. Takahashi and Yamanaka (2006) revealed that four transcription factors (Oct4, Sox2, c-Myc and Klf4) were sufficient to reprogramme mouse fibroblasts and other terminally differentiated cells into iPSCs (induced pluripotent stem cells). iPSCs have characteristics similar to ESCs, including the capability of self-renewal, large-scale expansion and differentiation towards all three germ layers. Moreover, early-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, suggesting that iPSCs are the potential replacement source for directed differentiation and clinical application of ESCs (Kim et al., 2010; Polo et al., 2010).

Meanwhile, the research into the pathways leading to lineage specification of ESCs indicate that Wnt, Notch and Hedgehog signalling pathways, which control many developmental processes are involved in ESC haemopoietic differentiation and haemopoietic progenitors' self-renewal (Reya et al., 2001; Rattis et al., 2004). Wnt3a, a member of the Wnt family of lipid-modified proteins, has been proved to initiate the transition from mesoderm to blood fate in mouse EB (embryoid body) cells through the canonical Wnt signalling pathway (Lengerke et al., 2008). But the manner of Wnt3a involvement in haemopoietic differentiation of iPSCs remains unclear and the haemopoietic stages affected by Wnt3a are also not well defined. Here, we employed an OP9 cell co-culture system to examine the effect of Wnt3a during different stages of haemopoietic differentiation of mouse ESCs and iPSCs. By analysing the percentage of Flk1+ and CD41+ cells, we have shown that Wnt3a is involved in the initiation of definitive haemopoiesis in both mouse ESCs and iPSCs.

2. Materials and methods

2.1. Cell lines and plasmids

TC-1 mESCs (mouse ESCs) and IP14D miPSCs (mouse iPSCs) (Zhao et al., 2009) were cultured on irradiated mouse embryonic fibroblasts in knockout DMEM (Dulbecco's modified Eagle's medium; Invitrogen) supplemented with 15% FBS (fetal bovine serum; Hyclone), 0.1 mM NEAA (non-essential amino acids; Invitrogen), 0.1 mM 2-mercaptoethanol (Sigma), 1 mM l-glutamine (Invitrogen) and 500 pM LIF (leukaemia inhibitory factor; Chemicon). Both miPSCs and mESCs were used between passages 10–20. pBPLV-Wnt3a plasmid was constructed using gene cloning and packaged in 293FT cells following the manufacturer's instructions (Invitrogen). Wnt3a-producing OP9 cells (named as Wnt3a-OP9 cells) were sorted and purified according to the expression of GFP (green fluorescent protein) and cultured with basic OP9 cell culture medium.

2.2. Differentiation of miPSCs and mESCs in co-culture with Wnt3a-OP9 cells

Undifferentiated and confluent miPSCs and mESCs were harvested using 0.05% (w/v) trypsin. Hanging drops were used to induce EB formation. Then 15 μl drops of cell suspension were densely plated on to the bottoms of 35-mm non-adherent Petri dishes via a micropipette; the plates were gently flipped over to invert the drops. Two days later, EBs were harvested, resuspended and plated into six-well dishes, either with irradiated Wnt3a-OP9 cells or control OP9 cells, in differentiation medium without additional factors. The cells were collected on days 3.75, 5 and 7.5 and trypsinized into single cells. Expression of Flk1, CD41 and c-kit/Sca-1 was detected using flow cytometry.

2.3. Antibodies and flow cytometry analysis

Differentiated cells were dissociated into single cells before washing and staining with FACS medium consisting of PBS supplemented with 2% FBS. The single cell suspension from day 3 was stained with either isotype control or Flk1–PE (phycoerythrin) antibodies (BD Pharmingen), while CD41–PE (BD Pharmingen) as well as Sca-1–PE, c-kit–FITC antibodies (BD Pharmingen) was used for days 5 and 7.5 co-cultured cells. All samples were analysed on a FACS Canto II flow cytometer (BD Biosciences). Other CD41-positive or -negative cells were used for Wright–Giemsa staining.

2.4. Gene expression analysis

Cells were dissolved using TRIzol® for total RNA isolation, employed as previously described (Takahashi et al., 2007). cDNAs were prepared with the oligo(dT) primer according the manufacturer's instructions (Takara). PCR was carried out using LATaq (Takara). Primers are listed in Supplementary Table S1 (at http://www.cellbiolint.org/cbi/036/cbi0360267add.htm).

3. Results

3.1. miPSCs and mESCs show equal potential EB formation

To characterize the differentiation potential of miPSCs, EBs were obtained from both TC-1 mESCs and IP14D miPSCs in hanging drops (Figure 1A). The EBs derived from mESCs and miPSCs exhibited similar 3D (three-dimensional) morphology. However, IP14D miPSC-derived EBs were initially smaller compared with TC-1 mESC-derived EBs (Figure 1B). In the miPSC-derived EBs, the expression of GFP was visibly decreased along with the time of in vitro culture, suggesting that IP14D miPSC-derived EBs were in the differentiation state (Figure 1C). Gene expression profiling of developing mESC- and miPSC-derived embryonic bodies demonstrated decreased expression of pluripotent genes of undifferentiated mESCs and miPSCs, including Oct4 and Sox2, accompanied by increased expression of the mesodermal genes, such as brachyury and Gata1 (GATA-binding protein 1). This indicates that mESC- and miPSC-derived EBs have the potential for haemopoietic differentiation.

3.2. miPSC- and mESC-derived CD41+ cells were significantly enriched in Wnt3a-OP9 cells co-culture system

A lentiviral vector containing Wnt3a gene named pBPLV-Wnt3a (Supplementary Figure S1A available at http://www.cellbiolint.org/cbi/036/cbi0360267add.htm) was constructed and packaged in 293FT cells. The virus products could be visualized by examining the expression of GFP (Supplementary Figure S1B). Wnt3a-OP9 cell line was generated by infecting OP9 cells with LV-Wnt3a virus. One week later, the cells were sorted according to the GFP expression levels (Supplementary Figure S2A available at http://www.cellbiolint.org/cbi/036/cbi0360267add.htm). The expression of Wnt3a gene was detected by RT–PCR (reverse transcription–PCR). The results showed that LV-Wnt3a-infected OP9 cells expressed high level of Wnt3a mRNA (Supplementary Figure S2B).

The mESC- and miPSC-derived EBs were accordingly co-cultured with Wnt3a-OP9 or OP9 cells. EBs attached to the stromal cells firmly after a day; the differentiation medium was changed every 2 days. Both mESC- and miPSC-derived EBs changed their morphology after co-culturing for 5 days, revealing a blast-like population (Figure 2A). Unlike spontaneous differentiation without any key factors, when seeding the one-day EBs on to the Wnt3a-OP9 cells, a visibly decreased expression of GFP could be detected from day 3 (Figure 2B). We collected cells by picking up the colonies under the microscope without disturbing stromal cells, and counted cells in the supernatant that display early haemopoietic cell characteristics.

The results of flow cytometry showed that the percentages of Flk1+ and CD41+ cells of OP9 co-cultured mESCs in our study were consistent with other studies (Mikkola et al., 2003). However, when co-cultured with Wnt3a-OP9 cells, the expression of Flk1 which was detected by flow cytometry on day 3.75 showed a significant stimulating effect of Wnt3a on both mouse ESCs and iPSCs' early stage haemopoietic differentiation (Figure 3A). The multiple experiments results demonstrated that the expression of Flk1+ cells were 47.11±2.61% of miPSCs-OP9 and 50.04±1.71% of miPSCs-Wnt3a-OP9 cells, 22.08±1.72% of mESCs-OP9 and 31.90±1.25% of mESCs-Wnt3a-OP9 on day 3.75. The same effect of Wnt3a was also found in the expression percentage of CD41+ cells, which was increased with a lower efficiency (Figure 3B). The rate of CD41+ cells were 1.53±0.29% of miPSCs-OP9 and 4.53±0.24% of miPSCs-Wnt3a-OP9 cells, and 1.29±0.19% of mESCs-OP9 and 2.76% ±0.13 of mESCs-Wnt3a-OP9 on day 5.

Morphologically, the results of the Wright–Giemsa staining showed that both miPSC- and mESC-EB derived CD41+ cells comprised a homogenous, blastocyst-like population (Figure 3C). To examine their haemopoietic potential, differentiated cells were plated into methylcellulose medium supplemented with a combination of haemopoietic growth factors. After 8 days of incubation, enriched CD41+ cells were found in definitive haemopoietic blastocyst cells, but with low efficiency, as most of the induced cells showed no growth or formed secondary EBs.

4. Discussion

iPSCs were first generated in mice in 2006 by Shinya Yamanaka and, shortly afterwards, successfully derived from human cells using analogous protocols. During the following 4 years, iPSC lines have been obtained, using different systems, from many kinds of cells (Okita et al., 2007; Yu et al., 2007; Park et al., 2008). iPSCs share many characteristics with ESCs, such as self-renewal, large-scale expansion and differentiation. When re-injected into the embryo, iPSCs chimerize all tissues, including the germ line. We focused on the role of Wnt3a in the early stage haemopoietic differentiation of mouse ESCs and iPSCs. A Wnt3a gene-modified OP9 cell line was established, which was used as the stromal cells to induce haemopoietic differentiation of mouse ESCs and iPSCs. Our results indicated that Wnt3a promoted the primitive haemopoietic differentiation of these pluripotent cells.

As a member of the Wnt gene family, Wnt3a is commonly characterized as potent morphogen, which can activate not only the canonical Wnt signalling pathway but also the non-canonical Wnt signalling pathway based on the cell or tissue type (Austin et al., 1997). By supplying Wnt3a during the in vitro differentiation of ESCs, one study showed that Wnt3a caused a notable increase in cell proliferation at all stages (Corrigan et al., 2008). This indicates that this growth factor plays an important role in regulating stem cell fate, including maintenance of pluripotency in mouse and human ESCs as well as the self-renewal of undifferentiated adult stem cells. In somatic reprogramming, Wnt3a is proved to promote the generation of iPSCs in the absence of the oncogene c-Myc by activating target genes through Wnt-β-catenin pathway. Besides, another downstream key transcriptional regulator of the Wnt pathway, Tcf3, also occupies and regulates the promoters of Oct4, Sox2 and Nanog (Cole et al., 2008; Tam et al., 2008), which also explains the effect of Wnt3a during the reprogramming. Research on human ESC differentiation suggests that co-operation of Wnt3a with BMP4 (bone morphogenetic protein 4) promotes haemopoietic differentiation of human ESCs (Reya et al., 2003). As a tightly controlled growth factors, Wnt3a can only been detected in two phases in haemopoietic differentiation of human ESCs, which corresponded to a period of the first definitive haemopoietic stem cell formation in the AGM (aorta–gonad–mesonephros) region, and at a later stage when LT-HSC (long-term haematopoietic stem cell) expansion occurs in the liver (Corrigan et al., 2008). In vivo studies also reveal that Wnt3a homozygous mutant embryos die around embryonic day (E) 12.5 of the embryonic development, further demonstrating that Wnt3a is involved in haemopoiesis of fetal liver.

We found in the early stages of haemopoietic differentiation, the percentage of Flk1+ and CD41+ cells were significantly increased when Wnt3a was added. This might be due to the effect of Wnt3a on increasing in both EB and haemopoietic colonies formation by the canonical Wnt signalling pathway. In addition, a growing body of evidence shows that the non-canonical pathway also plays an important role in haemopoiesis, such as Wnt5a and Wnt14 (Murdoch et al., 2003). Although current studies have shown an expression pattern of Wnt3a during definitive haemopoiesis, our work might prove that Wnt3a is also involved as an enhancer in the early stage of both mESC and miPSC differentiation, but the exact pattern of Wnt3a involvement remains unclear. There is also little doubt that further research is needed before ESCs/iPSCs can be used in a clinical setting.

Author contribution

Wencheng Zhang carried out the experiments and wrote the paper. Hailei Yao analysed the data and edited the paper. Sihan Wang and Shuangshuang Shi performed the flow cytometry. Yang Lv, Lijuan He and Xue Nan provided mESCs/iPSCs and feeder cells. Wen Yue participated in editing the article. Yanhua Li and Xuetao Pei were responsible for the study.

Acknowledgments

We thank Jie Hao and Xiaoyang Zhao from the State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, for cells and technical support.

Funding

This work was supported by the Major State Basic Research Program of China [grant numbers 2009CB941102 and2011CB964804] and the Project of Beijing Municipal Science & Technology Commission [grant number D07050701350702].

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Received 30 October 2010/22 August 2011; accepted 17 October 2011

Published as Cell Biology International Immediate Publication 17 October 2011, doi:10.1042/CBI20100766


© The Author(s) Journal compilation © 2012 Portland Press Limited


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)