Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Summary

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

Abstract

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Introduction

During early embryogenesis, gametogenesis occurs through a series of stages including primordial germ cell (PGC) formation, migration, and finally colonization of the gonadal ridges. During this time PGCs undergo proliferation and differentiation into increasingly more mature gametes ¹. The fact that PGCs migrate from the base of the allantois into the embryo hindgut and finally along the dorsal wall eventually colonizing the gonadal ridges makes them exceedingly difficult to study ². Despite advances in the field, studies attempting to understand PGC formation and differentiation have been impeded by their limited number, location, and migratory nature ^3,4.

In recent years embryonic stem cells have been shown to have the potential to form germ cells in vitro ^5,6. Similarly, several somatic stem cells have also been shown to have the potential to form germ cells following in vitro culturing ^7-11. Recently stem cells isolated from newborn mouse skin were differentiated in vitro into germ-like cells and early stage oocytes ¹². During differentiation the subset of the stem cells expressing Oct4 increased and structures resembling cumulus-oocyte complexes were formed. The oocyte-like cells (OLCs) can be picked from the cultures and compared to natural oocytes. Using this culture method OLCs measuring 40-45 μm are obtained that express similar markers to oocytes such as Gdf9b, VASA, and DAZL. To date the resulting OLCs remain unable to mature or be fertilized ¹².

Although the OLCs remain unable to function the protocol used to form these OLCs may still have utility as an assay to study the formation and development of earlier stage germ cells within a closed in vitro system. The original publication discussing the formation of OLCs from somatic stem cells utilized fetal porcine skin as a stem cell source ⁸. Expanding on that study the PGC-like cells formed early during induced differentiation were shown to be the precursors to the OLCs and express such PGC markers as OCT4, VASA, STELLA, C-KIT, and DAZL ¹³. This data supports the potential of utilizing this system to study germ cell formation and differentiation in vitro. The protocol utilized to form OLCs from newborn mouse skin will be demonstrated in this video article. This protocol offers an in vitro model to study germ cell development that may provide a means to elucidate factors important for their proliferation and differentiation.

Protocol

1. Media Preparation

1. Prepare media 2-3 hr in advance of use and place with a loosened cap in the cell culture incubator where the experiment will be taking place. Media used in the present protocol:
   1. Prepare stem cell medium consisting of DMEM/F12 supplemented with 1 x B27, 40 ng/ml bFGF, and 20 ng/ml EGF.
   2. Prepare germ cell differentiation medium consisting of m199 supplemented with 0.05 IU FSH, 0.03 IU LH, 3 mg/ml BSA, 5 μl/ml ITS, 0.23 mM sodium pyruvate, 1 mg/ml Fetuin, and 1 ng/ml EGF. A base medium consisting of all additives but omitting FSH, LH and EGF can be prepared and stored at 4 °C for up to one week.

2. Stem Cell Culture Preparation

1. Isolate stem cells from newborn mouse pups as previously described¹². Utilize stem cells at passage 2-4 for in vitro germ cell differentiation. The efficiency of OLC formation is directly related to the quality of the starting stem cells. It is best to start the differentiation as soon as the population appears clean and healthy which is generally at around passage 2. Further culturing the stem cells past passage 2 negatively affects the OLC formation efficiency (unpublished results).
2. 48 hr prior to initiating differentiation sub-culture the stem cells. Remove all the suspended spherical cell aggregates and spent media from the culture dish, using a serological pipette, and place in a 15 ml tube. It is important to only collect the suspended spherical aggregates of cells and not the cells attached to the bottom of the dish that are spontaneously differentiating.
3. Pellet the cells at 500 x g for 5 min., discard the supernatant, and resuspend in 500 μl of fresh stem cell media warmed to 37 °C. Gently pipette the aggregates to partially dissociate the cells. Do not aggressively pipette the cells as this will reduce cell viability. Wash the partially dissociated cells into 9.5 ml of fresh pre-warmed stem cell medium on a low attachment 10 cm cell culture dish.
4. Return the cells to a 37 °C, 5% CO₂ cell culture incubator for 48 hr prior to initiation of differentiation.

3. OLC Differentiation

1. Using a serological pipette remove the stem cells from the culture dish and place in a 15 ml tube, leave behind any attached cells. Pellet the cells at 500 x g and resuspend in 500 μl sterile PBS. Vigorous pipette the cells using a wide bore 1,000 μl tip into clumps not larger than 10-20 cells. Periodically remove a small amount of cells, during dissociation, and check under a white light microscope to confirm aggregated cells are in clumps of 10-20 individual cells. Over dissociation will result in reduced cell viability.
2. Add 9.5 ml PBS to the dissociated cells and add 15 μl of this cell suspension to a hemocytometer for cell counting. Pellet the cells at 500 x g for 5 min.
3. Re-suspend the cells at a concentration of 1.32X10⁶ cells/ml in differentiation medium.
4. Plate the cells by adding 500 μl per well into a flat bottom 24 well suspension dish.
5. Place the 24 well dish into a cell culture incubator at 37 °C in 5% CO₂ for 48 hr. Remove 250 μl medium from each well and place in a correspondingly labeled 1.5 ml tube. Add 250 μl fresh differentiation medium to each well. Centrifuge the spent medium at 500 x g for 5 min. and discard the supernatant. Resuspend any pelleted cells in 50 μl fresh differentiation medium and return to the corresponding culture well on the differentiation plate.
6. Every 48 hr change half the medium up to day 12 of differentiation.

Results

Initially the cells attach to the culture dish bottom and spread out. At 42-72 hr into differentiation small suspended OCT4 positive cells form and proliferate (Figure 1A). Shortly after the visualization of these cells the majority will disappear and the attached cells will form dense colony aggregates on the culture bottom. Following a few days these aggregates will detach from the culture surface. In some of these aggregates a large cell can be observed (Figure 1b and 1c

Discussion

To date functional OLCs have not been developed using a completely in vitro assay. Recently a study by Hayashi et al. was able to produce offspring from PGC-like cells formed in vitro and transferred in vivo for further development¹⁴. Starting with ES cells or induced pluripotent cells they were able to form PGC like cells which, when recovered following in vivo transplantation were able to be matured, fertilized, and used to generate offspring ¹⁴. Th...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM/F12	Invitrogen	12500
B27	Invitrogen	17504044
EGF	Invitrogen	PHG0311
bFGF	Invitrogen	PHG0266
ITS	Invitrogen	41400-045
BSA	Sigma	A-6003
Sodium Pyruvate	Invitrogen	11360-070
Fetuin	Sigma	F3385
FSH	Sioux Biochemicals	915
LH	Sioux Biochemicals	925
M199	Invitrogen	31100-035
24 well plates	Nunc	144530
10 cm dishes	VWR	25384-088
15 ml tubes	BD	352095