Page 1

Derivation of Clinical-grade Human Parthenogenetic ESCs and Graft of Their Progenitors in Parkinson’s Disease Monkey Models Qi Gu1,2, Jie Hao1, Wanwan Zhu1, Qi Zhou1,# 1. State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, PR China. 2. Intelligent Polymer Research Institute, ARC Center of Excellence for Electromaterials Science, AIIM Facility, University of Wollongong, Wollongong, New South Wales 2522, Australia Email: qg863@uowmail.edu.au, qizhou@ioz.ac.cn (# corresponding author)

Abstract

Methods

The cells differentiated from human embryonic stem cells (hESCs) are promising sources for cell therapy in regenerative medicine and for drug screening. However, most of the existing hESC lines are not available for clinical application due to safety and function issues (Gepstein, 2002). During application of the cells in therapeutic trials, all reagents used in the process, including feeder cells, culture media and equipments, should be tested and verified as safe according to good manufacturing practice (GMP) quality (Unger et al., 2008). Human parthenogenetic embryonic stem cells (hpESCs) possess great potential for applying histocompatible cells and tissues to cell therapy. Here, we report that we have generated one clinical-grade hpESC line (named chPES-1) that has been proved pluripotent and been qualified through sterility and pathogen testing. When the clinical-grade hpESC-derived dopaminergic neuron (DA) progenitors were grafted into the brain of MPTP-treated monkeys, the DA progenitors could survive and mature in vivo. This data suggested that our clinical-grade hpESCs can be used extensively in clinical therapy and research.

Parthenogenetic Inner cell mass (ICM) was isolated from the blastocyst embryos using mechanical method as previously described (Ström et al., 2007). The intact ICM was plated on Gamma ray-inactivated HFF feeder cells in NutriStem™ XF/FF Culture Medium. After culturing for 5-9 days, primary colonies would be seen and were passaged onto new feeder cells. At later passages, the ESCs were passaged by Collagenase NB6 (Nordmark Arzneimittel GmbH) as previously described (Crook et al., 2007). Figure 1 shows the overview of the whole procedure. Imunosurgery

EB

Incubation

DA

Passage

Graft

Figure 1

Results Figure 2 Pluripotent characterizations of chPES-1 cells OCT4

PI chPES-1

SOX2

SSEA4

PI

PI

D

Report

Gram staining

-

Endotoxin

-*

Mycoplasma

-

Amylase

Sox2

Ncstn

Nanog

Enolase

bovine virus

-

Lin28

Osteonectin

porcine virus

-

Gad1

Human T-lymphotropic Virus I

-

Human Immuno Deficiency Virus I

-

Human Immuno Deficiency Virus II

-

Human Hepatitis A Virus

-

Human Hepatitis B Virus

-

Human Hepatitis C virus

-

Human polyomavirus (JCV)

-

Human cytomegalovirus (HCMV)

-

TEM

-

Rex1 Gfap Gapdh

Gapdh

Endoderm

Virus Names

Oct4

Gdf3

A

Table 1 Sterility and pathogen testing

C

B

Mesoderm

Ectoderm

- indicated the test result negative * Endotoxin was tested negative <0.5EU/L

A Immunofluorescence analysis of chPES-1 cells. Clear expression of the ESCs surface antigen SSEA4 (green) and positive nuclear transcription factors OCT4 (purple) and SOX2 (green) were observed. DNA was stained by propidium iodide (red). B RT-PCR analysis of ES specific genes and confirm their expression. C Top, in vitro embryoid body formation. Bottom, endoderm (Amylase, Ncstn), mesoderm (Enolase, Osteonectin) and ectoderm (Gad1, Gfap) markers were detected by RT-PCR. D Teratoma formation. All three germ layers tissues were presented on the teratoma dissection slices identified by staining with haematoxylin and eosin. Scale bars, 500μm.

Figure 4 In vivo survival and maturation of chPES-1 derived DA progenitors in cynomolgus monkey Parkinson's disease models

A-B Cell grafts were traced by Prussian Blue staining for Fe visualization. A, Fe expression and graft morphology at 1 month after cell transplantation. B, some transplanted cells were found in the surrounding host tissue. C-E The grafts expressed TH (green), Rhodamine-B (red), Hochest33342(blue). F TH expression in migrated graft with surrounding host monkey tissue. G Expression of human specific nuclei and TH in the graft.

Figure 3 MIRB-labeling DA progenitor grafts can be traced by MRI scan A

B

A Red arrows point to the grafts in caudate and putamen region, with a big volume. B Red arrow points to the graft in substantia nigra region, with a small volume.

Conclusion

References

In our research, all reagents used were CTS™, Xeno-free or containing DMF. They were manufactured in the cGMP facilities and were provided with Certificates of Analysis and Certificates of Origin. Each of these precautions can be useful in applying for regulatory approval (Ausubel et al., 2011). The DA progenitors could survive in the Parkinson’s disease monkey brains have terminal differentiation. Production of clinical-grade stem cells will provide an important step in the possibility of stem cell therapy, and further animal model and pre-clinical testings will shed more light on the future use of these cells.

Ramos-Mejia, V., Rouleau, A., Yang, J., Bosse, M., et al. (2007). IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro. Nature 448, 1015-1021. Ellerstrom, C., Strehl, R., Moya, K., Andersson, K., Bergh, C., Lundin, K., Hyllner, J., and Semb, H. (2006). Derivation of a xeno-free human embryonic stem cell line. Stem Cells 24, 2170-2176. Unger, C., Skottman, H., Blomberg, P., Dilber, M.S., and Hovatta, O. (2008). Good manufacturing practice and clinical-grade human embryonic stem cell lines. Hum Mol Genet 17, R48-53. Schwartz, S.D., Hubschman, J.P., Heilwell, G., Franco-Cardenas, V., Pan, C.K., Ostrick, R.M., Mickunas, E., Gay, R., Klimanskaya, I., and Lanza, R. (2012). Embryonic stem cell trials for macular degeneration: a preliminary report. Lancet 379, 713-720.

Qi gu 2  
Read more
Read more
Similar to
Popular now
Just for you