Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model

Abstract

Background: To evaluate embryonic stem cell (ESC) harvesting methods with an emphasis on derivation of ESC lines without feeder cells or sera. Using a murine model, laser-assisted blastocyst dissection was performed and compared to conventional immunosurgery to assess a novel laser application for inner cell mass (ICM) isolation.

Methods: Intact blastocysts or isolated ICMs generated in a standard mouse strain were plated in medium with or without serum to compare ESC harvesting efficiency. ESC derivation was also undertaken in a feeder cell-free culture system.

Results: Although ICM growth and dissociation was comparable irrespective of the media components, an enhanced ESC harvest was observed in our serum-free medium (p < 0.01). ESC harvest rate was not affected by ICM isolation technique but was attenuated in the feeder cell-free group.

Conclusion: Achieving successful techniques for human ESC research is fundamentally dependent on preliminary work using experimental animals. In this study, all experimentally developed ESC lines manifested similar features to ESCs obtained from intact blastocysts in standard culture. Cell/sera free murine ESC harvest and propagation are feasible procedures for an embryology laboratory and await refinements for translation to human medical research.

Figure 1

Immunosurgery. Zona-free blastocyst (a) coated with rabbit anti-mouse antibodies. After addition of guinea pig complement, the outcome of unselective toxicity can be seen at t = 5 min (b), 15 min (c) and 30 min (d). Scale = 30 μm.

Figure 2

Laser dissection. Blastocyst secured by two holding pipettes with inner cell mass (ICM) being positioned at 9 o'clock before (a) and after (b) being sectioned by laser with (b) and without (c) zona pellucida. Arrows (b, c) indicate the resected area by laser energy. The smaller blastocyst fragment (white arrow) contains the ICM while the larger (yellow arrow) is exclusively trophoblast (d). Scale = 30 μm.

Figure 3

Grading embryonic stem cell colonies. Examples of ESC classification into one of the three grades: Grade Good (GG), Average (GA), or Poor (GP). In GG colonies the area occupied by pluripotent cells is >70% of the entire colony (a). Approximately 60% of the colony is occupied by pluripotent cells in GA colonies (b), and a GP colony features only ~20% of pluripotent cells (c). Scale = 30 μm.

Figure 4

ICM growth pattern as a function of media. Inner cell mass (ICM) growth patterns of intact blastocysts in standard DMEM medium (a-e) and in knockout culture system (f-j). Intact blastocysts plated on feeder cells (a, f). ICM attachment and trophoblast outgrowth (b, g). Multilayer differentiation of ICM on day 5 (D5) after plating, note endodermal "rind" (c). Immediately after first dissociation (d, i) blastocysts cultured in standard DMEM failed to produce ESC colonies (e) and generated epithelioid cells. In the knockout system, ICM growth showed negligible endodermal contamination (h) and after first dissociation with trypsin (red T bar) (i) generated ESC colonies (j). Scale = 50 μm.

Figure 5

Localization of pluripotency markers. Alkaline phosphatase (AP) activity (left), Oct-4 (center), and TROMA-1 (right) expression as measured in expanded and hatched blastocysts (top row, a-c), mouse embryonic fibroblasts (middle row, d-f) and embryonic stem cell colonies (bottom row, g-i) by immunofluorescent analysis. In left column, red signals indicate positive AP activity (a, g). In center, red signals identify positive Oct-4 expression (b, h) while the right column TROMA-1 expression is shown as green signals (c, hatched blastocyst). DAPI was used for nuclear DNA counterstaining as indicated by blue signals. Scale = 30 μm.

Figure 6

Coalescence and development of embryoid bodies. Day 1: Aggregation of individual ESCs to form spherical bodies (EB = embryiod body). Day 3: EBs ranging from 100–250 μm mean diameter. Day 4: Growing EBs of ~350 μm. Day 9: An EB with a cystic structure. Day 17: EB with outgrowth and vesicular cavity.

Figure 7

Ultrastructure of embryoid bodies. Transmission electron microscopy of embryoid body showing three embryonic germ layers including keratin filaments (ectoderm), cardiomyocyte with contractile components (mesoderm), and microvilli (endoderm).

Figure 8

Histology of teratoma induced by ESC injection. Teratoma was induced by injection of ESC into SCID mouse testis. Ectodermal differentiation is observed with basophilic staining in a cord-like distribution, typical neural tissue organization (a1), and neuronal rosette (a1 inset). Squamous epithelium shows normal keratin deposition pattern (a2). Mesodermal components include cartilage (b1) and striated muscle (b2). Endodermal differentiation to a gut-like structure lined with mucinous epithelium (c1). Cells organized in acinar structure typical of glands (pancreas or salivary) (c2).

Figure 9

Organization of primitive endoderm. Schematic of expanded blastocyst with absence (a) and presence (b) of primitive endoderm (hypoblast) in a day 4 expanded blastocyst and day 6 hatched blastocyst, respectively. In b, ICM remnant is defined as the epiblast (green) and the hypoblast (yellow). Hatching blastocyst (c) with epiblast (green arrow) and hypoblast (yellow arrow). Scale = 30 μm.

Figure 10

Vesicle formation post-immunosurgery. Unilaminar vesicle generated from plated ICM after immunosurgery with obvious endodermal contamination at day 1 (a) and day 2 (b). Scale = 30 μm.

Figure 11

Observed growth patterns according to ICM isolation technique. Attachment of ICM and trophectodermal outgrowth with large (b1, b2, after immunosurgery) or minimal (a1, a2, intact blastocyst) endodermal contamination. Such contamination is completely absent following laser dissection (c1, c2). First column shown at 200×, second column shown at 400×. Scale = 50 μm.