Induction of embryonic stem cells to hematopoietic cells in vitro

XU ~ing', QlAO chunping2, HUANG ~haoliang', LI shunong2 8 WU xuan3 1. Department of Pediatrics, Sun Yat-sen Memorial Hospital. Sun Yat-sen University of Medical Sciences, Guangzhou 510120.

China; 2. Department of Pathophysiology, Sun Yat-sen University of Medical Sciences, Guangzhou 5 14089, China; 3. Department of Physiology, the Third Military Medical University, Chongqing 400038, China Correspondence should be addressed to Wu Xuan (e-mail: twx6903@mail.tmmu.com.cn)

Abstract In order to get hematopoietic cells from embryonic stem (ES) cells and to study development mechanisms of hematopoietic cells, the method of inducing embryonic stem cells to hematopoietic cells was explored by differenciating mouse ES cells and human embryonic cells in three stages. The differentiated cells were identified by flow cytometry, immunohistochemistry and Wright's staining. The results showed that embryoid bodies (EBs) could form when ES cells were cultured in the medium with 2-mercaptoethanol (2-ME). However, cytokines, such as stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), interleukind (IL-6), erythropoietin (EPO) and granular colony stimulating factor (G-CSF), were not helpful for forming EBs. SCF, TPO and embryonic cell conditional medium were useful for the differentiation of mouse EBs to hematopoietic progenitors. Eighty-six percent of these cells were CD34' after 6-d culture. Hematopoietic progenitors differentiated to B lymphocytes when they were cocultured with primary bone marrow stroma cells in the DMEM medium with SCF and IL-6. 14 d later, most of the cells were CD34-CD38'. Wright's staining and immunohistochemistry showed that 80% of these cells were plasma-like morphologically and immunoglubolin positive. The study of hematopoietic cells from human embryonic cells showed that human embryonic cell differentiation was very similar to that of mouse ES cells. They could form EBs in the first stage and the CD34 positive cells account for about 48.5% in the second stage. Keywords: embryonic stem cell, cytokine, hematopoietic stem cell.

The embryonic stem cell line (ES cells) from mouse, chicken, rabbit, pig and rhesus have been established after Evans et al. established the first mouse ES cell line in 1981. In January of 1998, we reported the isolation and culture of human embryonic cells[61. The cells were cultured for 6 human generations, maitaining undifferentiated. In November of 1998, ~homson[" pronounced in Science that human ES cell line was established.

ES cells can differentiate to various cells in vitro, including neurons, cardiomyocytes and hematopoietic cells. Many scientists focus on how to get the pure specific cells, because it is very useful not only for supplying the materials for transplantation, but also for studying the mechanisms of cell differentiation and development, especially for gene regulation in vitro. In order to supply the hematopoietic cells to clinical diagnosis and to study the mechanisms of hematopoietic cell development, the method for differentiating mouse ES cells and human embryonic cells to hematopoietic cells was studied in the present study.

1 Materials and methods

( i ) Materials. ES-D3 cells and Buffalo rat liver cells (BRL) were provided by Prof. Cong Xiaoqian of the Institute of Shanghai Cell Biology, the Chinese Academy of Sciences. Other materials were fetal bovine serum (Hyclone Co., USA), ABC kit (Fuzhou Maixin Co., China), FITC-conjugated rat anti-mouse CD34, PE-conjugated rat anti-mouse CD38 and PE-conjugated rat-human CD34 (Pharrningen Co., USA), recombinant human stem cell factor (SCF), recombinant human interleukin-3 (IL-3), granular colony stimulating factor (G-CSF) (Kirin Pharmaceutical Co., China), thrombopoietin (TPO) (Hangzhou Jiuyuan Gene Co., China), and Kunming mouse (Animal Center of the Sat-Yet-sen University of Medical Sciences).

( ii ) Cell culture. (1) Maintenance of ES-D3 cell line and culture of mouse primary embryonic fibroblast cells.

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They are described in ref. [8]. (2) BRL cell culture and BRL conditional medium preparation. BRL cells were plated at a cell

density of 2X 10~lmL in Dulbecco's modified eagle's medium (DMEM)-1OZFCS medium and grown on the tissue culture surface. 72 h later, the cell-free supernatants were collected by centrifugation, filtrated with a 0.22-ym filter, and then 7.5% FCS was added to the filtrated medium to get solution A, which was mixed with ES-D3 culture medium (B) at a ratio (Am) of 60140 (VIV). Finally, this medium was supplemented with 2-mercaptoethanol(2-ME). The final concentration was 0.1 mmol L-I.

(3) Preparation of primary bone marrow stroma cells. Bone marrow of mouse femur was flushed out with a 1-mL syringe. Bone marrow cells were washed twice by DMEM, collected with centrifugation and then plated on dishes, the culture medium was a-MEM-15%FCS. These cells must be pretreated by rnitomycin for 1 h before use as feeder layer.

(4) Preparation of embryonic cell conditional medium. ES-D3 cells were plated at a cell density of 5 X 104/rn~ in Iscove's modified Dulbecco's medium (IMDM) with FCS (15%) and 2-ME mol L-I). Three days later, the cell-free supernatants were collected and filtrated. The concentration of conditional medium was 25% in the medium for induction.

(5) The isolation and culture of human embryonic cell. See ref. [6]. (iii) Induction of mouse ES-D3 cells to hematopoietic cells by stages. In stage 1, ES-D3 cells

grew at a cell density of 1 X 1 0 4 / m ~ in IMDM-15% FCS supplymented with 0.9% methylcellulose. These cells were divided into 5 groups as described in table 1. There were 3 wells in each group. These media were supplemented by different combinations of 2-ME (10-'mol L-I), SCF (50 ng m.L-I). EPO (4 u rn~-I), TPO (100 ng mL-I), IL-3 (50 ng mL-I), IL-6 (50 ng K') and G-CSF (100 ng m ~ - ' ) as described in table 1. 3.5 d later, cells were observed under an invert-microscope. This step was repeated 3 times.

Table I Effect of cytokines on the first-stage differenciation of ES-D3 cells Group Cytokine Result (on the 3.5th day)

1 SCF, TPO, IL-6, IL-3, EPO, G-CSF cells died 2 SCF cells grew slowly, no EB 3 IL-6 cells grew slowly, no EB

4 2-ME EBs formed

5 2-ME, CM no EB

CM, Conditional medium; EB, embryoid body.

In stage 2, embryoid bodies were collected by centrifugation, dissociated by 0.05% trypsin and replated on dishes in 3 groups. Different combinations of SCF (50 ng m ~ - I ) , TPO (50 ng mL-I) and 2-ME (10-'mol L-') were added to the three groups of media (table 2).

Table 2 Effect of cytokines on the second-stage ES-D3 cells differentiation

Cytokine Result (on the 4th day)

SCF, TPO growing slowly

SCF, TPO, 2-ME most cells sticking on the wall

SCF, TPO, CM forming hematopoietic-like cell colonies

In stage 3, cells were collected by centrifugation and plated onto bone marrow stroma cells at a cell density of l x l ~ ~ / m ~ . SCF (50 ng mL-') and IL-6 (50 ng mL-') were added to the medium. Cells were collected after 6 d and detected by Wright's staining, flow cytometry and immunohistochemistry.

(iv) Induction of human embryonic cells to hematopoietic cells. Based on the induction of mouse ES-D3 cells to hematopoietic cells, the same method was used, but the time for each stage was prolonged.

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2 Results

( i ) Induction of hematopoietic cells from ES-D3 cells by three stages. (1) Formation of embryoid bodies. The cells of the first group which were cultured in

methylcellulose with 2-ME, SCF, EPO, TPO, IL-3, IL-6 and G-CSF were dead and the medium became cloudy due to the large number of dead cells. The possibility of contamination or high density of cells was excluded (cell density was 1 X 10~1well). Cells of the third group which were cultured in methylcellulose with SCF grew slowly with few colonies and no embryoid bodies were found, while the cells of the fourth group which were cultured with 2-ME grew well with embryoid bodies. The cells of the fifth group which were cultured with conditional medium and 2-ME grew not well (table 1).

The results suggested that cytokines are not required at the early stage of ES-D3 cell development for the formation of embryoid bodies.

(2) Differenciation of ES cells to hematopoietic cells. In the second stage (table 2), the cells cultured in the medium with SCF and TPO grew slowly. 2-ME could facilitate the growth of cells, but most cells stuck on the wall, like endothelial cells. Most of the cells can form cell colonies when conditional medium was added (fig. 1). Flow cytometry detecting on the 6th day differentiation showed that 86% of these cells were CD34'.

Fig. 1 . Induction of mouse ES cells to hematopoietic cells. (a) The 3.5-d EB from mouse ES-D3 ( ~ 2 5 0 ) . (b) Non-sticking cell colonies from mouse ES-D3 ( ~ 1 0 0 ) . (c) Plasma cells from mouse ES-D3 (Wright's staining) ( x l 000). (d) Immunoglobulin staining of plasma cells induced from mouse ES-D3 cells (immunohistochemistry staining) (xl 000).

(3) Differenciation to plasma cells. After 7 days, cells of the third group were plated on primary bone marrow stroma cells in DMEM with SCF and IL-6. On the 14th day, most cells showed CD34-CD38' and 80% cells were morphologically like plasma cells, which were characterized by side- drifting of nuclear cells, plenty of cytoplasma, and less ratio of nucleus to cytoplasma (fig. l(c)). The immunoglobulin staining was positive with brown staining in cytoplasma (fig. l(d)).

( ii ) Induction of human embryonic cells to hematopoietic cells by three-stage culture.

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(1) Formation of embryoid bodies. The embryoid bodies formed when embryonic cells were cultured in methylcellulose with 2-ME and the embryoid bodies were about 10 times larger than embryonic cells (fig. 2(a)).

Fig. 2. Induction of human embryonic cells to hematopoietic cells. (a) EBs from human embryonic cells (x40). (b) Hematopoietic-like cell colonies from human embryonic cells ( ~ 1 0 0 ) .

(2) Embryonic cells differentiate to hematopoietic progenitors. Similar to the differentiation of mouse ES cells in the second stage, most cells could form cell colonies consisting of small and erythrocyte-like cells (fig. 2(b)), while some cells stuck on the wall and grew like endothelial cells. The CD34' cells were increased by 48.5% after 14-d culture (fig. 3).

0 200 400 600 800 1000 0.1 1 10 100 1000 FITC FlTC

Fig. 3. CD34 expression of the induced cells increased on the 14th day. (a) CD34: 8.7%. (b) CD34: 57.2%. FITC means fluorescein isothiocyanate. A in fig. 3(b) stands for the positive range.

3 Discussion

( i ) Induction of mouse ES-D3 cells to hematopoietic stemlprogenitor cells in vitro. Embryonic stem cells were derived from totipotent cells of early embryo and capable of unlimited, undifferentiated proliferation in virro. They have the potential of differentiating to any kind of cells or tissues.

Mouse ES cells have broad potential of differentiation to various hematopoietic cells, such as erythroid, granulocyte/macrophage, megakaryocyte, mast and lymphocyte precursors. It was reported by ~alacios '~ ' that the cell-sorter-purified pgP-l+ line-cells, which were differentiated from ES cells by coculturing with the stromal cell line RPOlO in the medium with the combination of interleukin-3, interleukin-6 and F (cell-free supernatants from cultures of the FLS 4.1 fetal liver stromal cell line), could repopulate the lymphoid, myoloid and erythroid lineages of irradiated mice.

In the early stage of our research, we cultured ES cells directly on the feeder layer combined with different cytokines as described before. It was found that cells did not grow very well or were induced to a mixture of various cells, such as hematopoietic cells, endothelial cells and cardiomyocytes. The ideal results were obtained when we divided the process into three steps, during which several different microcircumstances were provided in accordance with the different development stages. At the first

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PAPERS stage, 2-mercaptoethanol (2-ME) was added into the medium to help embryoid body formation. Cytokines were not needed at this stage. It was reported['" that 2-ME prevente the loss of glutathine that occurs when cells are cultured and that 2-ME improved the proliferation capacity of cells. Cytokines were not needed because the receptors of cytokine were not expressed on the membrane of embryoid body cells.

At the second stage, in response to SCF, TPO and conditional medium, embryoid bodies differentiate to hematopoietic cells. It has been reported that SCF and TPO could promote the proliferation and survival of bone marrow stem cells. In the present study, using SCF and TPO only made embryoid body cells differentiate very slowly. The conditional medium improved the proliferation of embryoid body cells and gave rise to colonies that express CD34. These cells were small and similar morphologically to the early stage hematopoietic cells. These results indicated that the combinative use of conditional medium may contribute a lot to the differentiation of ES cells to hematopoietic cells. ~ e n n e d ~ ' " ] reported that D4T CM medium conditioned by an EB-derived endothelial cell line promoted the growth of cells at this stage, which coincided with our results. The conditional medium propably contained factors secreted by embryonic cells which can promote the differentiation of mesoderm-derived cells to hernatopoietic cells. SCF and its receptor were expressed during the differentiation of ES, and SCF could improve the proliferation and survival of bone marrow hemato oietic stem cells and stimulate the differentiation of ES to hematopoietic cells. In the recent P studyr12, thrombopoietin (TPO) has also been proved capable of improving the survival of hema- topoietic stedprogenitor cells.

( ii ) Induction of human embryonic stem cells to hematopoietic stedprogenitor cells in vitro. The isolation and culture of human ES cells were reported in 1998[6.71. Successful induction of human ES cells to hematopoietic stemlprogenitor cells has not been reported yet. Based on the molecular level conservation of animal development regulation mechanism, the results of animal research help the human development research. The limitation of getting zygotes and the unestablishment of stable human ES cell lines made our research difficult. In the present study, human ES cells were induced by stages essentially as performed for mouse ES cell induction. It was found that embryonic cells derived from morula could differentiate to hematopoietic cells under a certain condition. Compared with the results from mouse ES cells, the induction of human ES cells needed longer time. Due to the limitation of zygotes, we did not research the problem whether 2-ME is indispensable for the formation of embryoid bodies or not.

Our results showed that human development shared the similar rule with mouse development. It is very important to refer to the research on mouse ES cells.

Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 39800134) and the Natural Science Foundation of Guangdong Province (Grant No. 980687).

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