In vitro female germline potential of human umbilical

In vitro female germline potential of human umbilical cord-derived matrix stem cells★

Li Cai-xia, Wang Feng-ying, Liang Zhi-qing, Li Yu-yan, Yu Chi-yang, Chang Qing, Long Ling Abstract

BACKGROUND: Bone marrow mesenchymal stem cells (BM-MSCs) have been shown to possess the potential to differentiate

into oocytes. However, immune rejection and a limited number of donors of BM-MSCs constrain the applications of BM-MSCs.

Several studies have demonstrated that human umbilical cord matrix stem cells (UC-MSCs) also have an intrinsic ability to

differentiate into oocyte-like cells in vitro.

OBJECTIVE: To establish the method for UC-MSCs culture and to investigate the in vitro differentiation potential of UC-MSCs

towards germ cells.

METHODS: Umbilical cord from full-term normal deliveries was obtained in sterile condition. Collagenase I-digested cells were

cultured in DMEM. The immunophenotype of cells was determined by flow cytometry. Lipoblasts, osteoblasts and chondroblasts

were induced in different condition cultures. The expression of germ cells specific marker in UC-MSCs was determined by reverse

transcription-polymerase chain reaction. Follicular fluid was employed to induce

UC-MSCs differentiation into germ cells.

RESULTS AND CONCLUSION: Spindle-like umbilical cord cells were shown and cells in culture were extended to more than 10

passages. BM-MSCs-like immunophenotypes were shown: CD29, CD44, CD73 (SH3), CD90 and CD105 (SH2) were positive;

SSEA-4 was weakly positive; CD31, CD34, CD45 and HLA-DR were negative. After

UC-MSCs were induced in different condition

cultures, lipid droplet-, bone tubercle-, and cartilage tubercle-like structures emerged and the mRNA expressions of specific gene

of fat, bone and cartilage were observed. Germ cells markers, OCT4, Stella, Ifitm3, were expressed in UC-MSCs. After induced

by 5%, 10% or 20% follicular fluid, cells aggregated and oocyte-like structures were observed. Human UC-MSCs could be

cultured and amplificated in vitro. UC-MSCs showed immunophenotypes similar to BM-MSCs. UC-MSCs had the potential to

differentiate into lipoblasts, osteoblasts, and chondroblasts. Oocyte-like structure was induced in vitro from UC-MSCs with germ

cells specific marker. These findings suggest that UC-NSCs have the potential to differentiate into germ cells.

NTRODUCTION

A central dogma of reproductive biology has been that

the postnatal ovary harbors a limited supply of

oocytes that cannot be replenished or regenerated if

lost during injury or disease [1]. However, this has been

challenged recently by the surprising observation that

embryonic stem cells (ESCs) and bone marrow

mesenchymal stem cells (BM-MSCs) can differentiate

into female germ cells[2-3] . These discoveries would

benefit the study of oogenesis and advance the field

of therapeutic cloning by providing an alternative

source of oocytes. However, immune rejection and a

limited number of cell donors[4-5] constrain the

applications of ESCs and BM-MSCs. Hence, there is

an acute need to find alternative cell sources for such

cell therapies.

One potential alternative source of MSCs appears

feasible with the report of the successful culture of human umbilical cord-derived stem cells[6]. Umbilical cord stem cells express low levels of human leukocyte antigen (HLA) and have been used for bone marrow replacement in case HLA-matched donors cannot be found [7]. Mesenchymal precursors have been isolated from umbilical cords and these cells can be induced to differentiate into lipoblasts, osteoblasts, chondroblasts or "neurocyte-like" cells[8-11] . Thus, we postulate that these cells would probably be a potential alternative source of mesenchymal stem cells to generate oocytes.

Here we isolated matrix stem cells from human umbilical cords (UC-MSCs) and observed the expression of some germ cell markers on these cells and oocyte-like structures. Our results support