Publication Date

2011

Document Type

Thesis

Committee Members

Lawrence Amesse (Committee Member), Yanfang Chen (Advisor), Mariana Morris (Committee Member), James Olson (Committee Member)

Degree Name

Master of Science (MS)

Abstract

Human endometrium is a high dynamic tissue that contains stem/progenitor cells. These endometrial stromal progenitor cells (hESCs) have been differentiated into a number of mesodermal lineages. There is limited information on differentiating hESCs into neurons, and no information on differentiating hESCs into megakaryocytes (MKs) and platelets (PLTs). The aim of this work was to investigate the possibility of differentiating hESCs into two distinct lineages: MKs, with subsequent PLT formation, and neurons.

We isolated hESCs from human endometrial tissue and cultured the cells for 4-6 passages. Before each differentiation experiment, the purity of hESCs was confirmed by flow cytometry analysis which revealed that the cells were highly positive (>95%) for CD90 and CD29 (stromal cell markers), and were negative (<1.3%) for CD45 and CD34 (hematopoietic cell markers).

For MKs differentiation, hESCs (1.8 X 105 cells/well) were cultured in serum-free medium either with (differentiation) or without (control) thrombopoietin (TPO, 50 ng/ml) for 18 days. MKs differentiation was analyzed by flow cytometry and confocal microscopy. PLTs were collected from culture medium supernatant from day10-18, and were defined by flow cytometry and functional study. Our results show: 1) MKs were successfully generated as evidenced by expression of MK membrane markers (CD41a: 39 ± 3.0% and 1 ± 0.09%; CD42b: 28 ± 2.0% and 1.2 ± 0.06%, differentiation vs. control, n=3). 2) Immunocytochemistry analysis showed the differentiation rate of CD41a (38 ± 3.0%) and CD42b (27 ± 2.5%). 3) Generated PLTs were positively labeled with CD41a (90 ± 2%). 4) Functional study of generated PLTs revealed that thrombin (5 U/ml) stimulation up-regulated CD62P expression (26.0 ± 4% and 2.5 ± 1%, thrombin vs. control, n=3), and fibrinogen binding (32 ± 3.0% and 1 ± 0.4%, thrombin vs. control, n=3). 5) Electronic microscopic examination showed that generated PLTs had similar ultrastructure (storage granules) as normal human peripheral blood PLTs.

For neuron differentiation, hESCs (1.6 X 105 cells/well) were cultured in medium either with (differentiation) or without (control) cytokines (FGF, EGF, NGF and BDNF) for 14 days. Differentiated neurons were analyzed by confocal microscopy and western blot analysis. Our results show: 1) neurons were successfully generated as evidenced by phenotypic expression of neuron specific markers nestin (40 ± 4%), β-tublin III (37 ± 3%), NeuN (15 ± 2%), TH (14 ± 1%), and astrocyte marker GFAP (10 ± 3%). 2) Western blot analysis showed that the expression of transcription genes (Oct4: 0.11 ± 0.01and 0.32 ± 0.01; Sox2: 0.25 ± 0.02 and 0.52 ± 0.03, differentiated vs. control, n=4) was down-regulated in differentiated neurons.

Taken together, these data indicate that it's feasible to generate MKs, functional PLTs and neurons from hESCs. The hESCs could be a potential source for cell-based therapy in regenerative medicine.

Page Count

68

Department or Program

Department of Pharmacology and Toxicology

Year Degree Awarded

2011

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