Skip to content

Advertisement

  • Research
  • Open Access

High-efficiency derivation of human embryonic stem cell lines using a culture system with minimized trophoblast cell proliferation

  • 1,
  • 1,
  • 2,
  • 2,
  • 2,
  • 2,
  • 2,
  • 3,
  • 4,
  • 1,
  • 1, 5,
  • 1,
  • 1, 6 and
  • 1, 5Email author
Stem Cell Research & Therapy20189:138

https://doi.org/10.1186/s13287-018-0866-5

  • Received: 7 February 2018
  • Accepted: 11 April 2018
  • Published:

Abstract

Background

Due to their extensive self-renewal and multilineage differentiation capacity, human embryonic stem cells (hESCs) have great potential for studying developmental biology, disease modeling, and developing cell replacement therapy. The first hESC line was generated in 1998 by culturing inner cell mass (ICM) cells isolated from human blastocysts using an immunosurgery technique. Since then, many techniques including mechanical ICM isolation, laser dissection, and whole embryo culture have been used to derive hESC lines. However, the hESC derivation efficiency remains low, usually less than 50%, and it requires a large number of human embryos to derive a significant number of hESC lines. Due to a shortage of and restricted access to human embryos, a novel approach with better hESC derivation efficiency is badly needed to decrease the number of embryos used.

Methods

We hypothesized that the low hESC derivation efficiency might be due to extensive proliferation of trophoblast (TE) cells which could interfere with ICM proliferation. We therefore developed a methodology to minimize TE cell proliferation by culturing ICM in a feeder-free system for 3 days before transferring them onto feeder cells.

Results

This minimized trophoblast cell proliferation (MTP) technique could be successfully used to derive hESCs from normal, abnormal, and frozen–thawed embryos with better derivation efficiency of more than 50% (range 50–100%; median 70%).

Conclusions

We successfully developed a better hESC derivation methodology using the “MTP” culture system. This methodology can be effectively used to derive hESCs from both normal and abnormal embryos under feeder-free conditions with higher efficiency when compared with other methodologies. With this methodology, large-scale production of clinical-grade hESCs is feasible.

Keywords

  • Human embryonic stem cells
  • Embryo
  • Clinical grade
  • Trophoblast cells

Background

Human embryonic stem cells (hESCs) have the capability to self-renew indefinitely in culture while retaining their ability to differentiate to all cell types. hESCs not only play important roles in basic research in developmental biology, disease pathogenesis, and gene function, but also serve as a valuable model for drug screening and tissue transplantation. At present, more than 300 hESC lines have been eligibly registered to the National Institutes of Health Human Embryonic Stem Cell Registration (http://grants.nih.gov/stem_cells/registry/current.htm). Most hESCs are usually derived from discarded embryos or embryos donated from a couple who have completed in vitro fertilization (IVF) treatments and have no desire to utilize the remaining embryos for transplantation [1, 2]. To generate hESC lines for various research and clinical applications, a large number of human embryos are usually required (Additional file 1: Table S1) [2, 3]. To derive hESCs, immunosurgery, mechanical ICM dissection (MID), and whole embryo culture (WEC) techniques are developed. Immunosurgery is a method for removing TE cells, the outer cell layer of blastocysts, through complement-dependent antibody cytotoxicity [4]. Because an animal-derived antibody is required for immunosurgery, this technique is therefore unsuitable for deriving clinical-grade hESCs. Exposing hESCs to animal products could increase risks of zoonosis and immune reaction against contaminated nonhuman molecules, such as sialic acid N-Glycolylneuraminic acid (Neu5Gc), resulting in the rejection of transplanted hESCs [3, 512]. Derivation of hESCs with either MID or WEC can be used to derive clinical-grade hESCs. However, the hESC derivation efficiency with those methods is generally low [1, 1316]. Due to a shortage of and restricted access to human embryos, a more efficient procedure for deriving hESCs under xeno-free conditions is critically needed.

Although induced pluripotent stem cells (iPSCs) generated by epigenetic reprogramming of adult somatic cells can be used as a substitute for hESCs for studying basic biology and pathophysiology of human diseases [17], several recent studies reported that many iPSC lines have acquired various genetic mutations during the course of their generation and expansion that might compromise their clinical use [1828].

In the present study, we successfully develop better a hESC derivation methodology using a culture system called “minimized trophoblast cell proliferation” (MTP). This methodology can be effectively used to derive hESCs from both normal and abnormal embryos under feeder-free conditions with higher efficiency when compared with other methodologies.

Methods

Ethical permission for human embryos used

The study protocol was approved by Siriraj Institutional Review Board, Faculty of Medicine, Siriraj Hospital (SIRB), Mahidol University (Si338/2013). All experiments were performed under the guidelines and regulations of SIRB, Mahidol University. The human embryos used in this study were obtained from the infertility unit, Siriraj Hospital. Informed consent was obtained from all couples that donated spare embryos following in vitro fertilization (IVF) treatment. Before giving consent, the couples were provided with all of the necessary information about the research project and they were made aware of the sensitive nature of the study. Only embryos with genetic abnormalities, diagnosed by performing blastomere biopsy and preimplantation genetic diagnosis (PGD), were used for embryonic stem cell derivation.

Derivation of human embryonic stem cells

Whole embryo culture method

The zona pellucida (ZP) was removed by incubating the embryos with 0.1% (w/v) pronase for 5 min followed by washing in NutriStem medium (Stemgent, USA). The ZP-free embryos were then cultured on human foreskin fibroblasts (HFFs) in NutriStem media under hypoxic conditions (a humidified atmosphere of 5%O2, 5%CO2, and 90%N2) (Fig. 1a, b).
Fig. 1
Fig. 1

Derivation of hESCs by WEC and MID. a Process of hESC derivation from human blastocysts by WEC and MID. b Derivation of hESCs by WEC: culture whole embryo on HFFs without ICM isolation. c, d ICM (arrowheads) mechanically separated from TE cells (arrow) by glass pipette (c) before transferring onto HFFs for further expansion (d). Scale bar: 50 μm. MID: mechanical ICM dissection, ICM: inner cell mass, WEC: whole embryo culture, HFFs: human foreskin fibroblasts

Mechanical ICM dissection method

The ZP-free embryos were transferred to the micromanipulator for ICM isolation. Firstly, the embryos were suspended in a micro-drop of NutriStem medium and their ICMs were adjusted to the 12 o’clock position. A fine glass needle was then pressed on top of the embryos in the region under their ICMs while the holding pipette was placed underneath the needle and moved back and forth several times to tear off TE cells (Fig. 1c). The isolated ICM was then cultured on HFFs in Nutristem medium under hypoxic conditions (Fig. 1a, d).

Minimized trophoblast cell proliferation method

The ICMs isolated by MID were cultured on plates coated with either CELLstart (Gibco, USA) or Matrigel (Corning, USA) in Nutristem medium under hypoxic conditions. After 3 days of culture, the ICM clump was mechanically separated from the surrounding TE cells and transferred to a fresh culture plate containing HFFs (Fig. 2b). At this stage, half of the culture medium was replaced by fresh medium every day throughout the entire culture period. The culture was observed daily and the cells were mechanically subcultured when their colonies reached optimal size.
Fig. 2
Fig. 2

Derivation of hESCs by MTP. a On day 2 after ICM isolation, prominent ICM clump (arrowheads) surrounded with proliferating TE cells observed. Under feeder-free conditions using either CELLstart or Matrigel, most proliferating TE cells were degenerated on culture day 3 (arrows) while remaining ICM clump was transferred to fresh HFFs for further expansion. b Procedure of hESC derivation by MTP method. Scale bar: 50 μm

hESC culture

hESC colonies were cut into small pieces with a fine glass needle and transferred onto freshly prepared gamma-irradiated HFFs. The cells were then cultured in NutriStem medium (Biological Industries, USA) under hypoxic conditions and were subcultured every 4–5 days when their colonies reached optimal size. For the feeder-free system, hESC colonies were cultured on a Matrigel-coated plate in NutriStem media under hypoxic conditions and subcultured every 4–5 days using Versene treatment (Life Technologies, USA).

Karyotype analysis

hESCs were expanded under feeder-free conditions until their density reached 100% confluence. At this stage, the cells were harvested and sent to Siriraj Central Cytogenetic Laboratory, Faculty of Medicine, Siriraj Hospital, Mahidol University for karyotyping.

Studying the expression of pluripotent marker genes by RT-PCR

Expression of OCT4, SOX2, and NANOG genes was determined by reverse transcription PCR (RT-PCR). Briefly, total RNA was isolated by Trizol Reagent (Invitrogen, USA), according to the manufacturer’s instructions, and 1 μg of RNA was converted to cDNA by the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). The PCR was then performed and the results were analyzed by agarose gel electrophoresis. The primer sequences are presented in Additional file 1: Table S3.

Immunofluorescence staining

Cells were fixed with 4% (w/v) paraformaldehyde in PBS and their membranes were permeabilized with 0.1% (w/v) Triton X-100 in PBS. At this stage, the cells were incubated with 3% (w/v) BSA in PBS at room temperature for 2 h to prevent nonspecific antibody reaction before incubating with mouse antibodies against human NANOG (1:100; Millipore), OCT4 (1:100; Santa Cruz), SOX2 (1:200; Millipore), SSEA4 (1:100; Millipore), TRA1–60 (1:100; Millipore), and TRA-1-81 (1:100; Millipore) at 4 °C overnight. After incubation with primary antibodies, the cells were washed twice with PBS and further incubated with appropriate secondary antibodies (1:500) at room temperature for 1 h. The nuclei were visualized by staining with Hoechst33342 (Life Technologies, USA) and the cells were studied by fluorescent microscopy.

In vitro differentiation

hESCs were harvested by incubation with dispase and transferred to low-adherent culture dishes (Corning, USA) containing DMEM (Invitrogen, USA) supplemented with 20% (v/v) serum replacement (Invitrogen, USA), 10 mM non-essential amino acids (Invitrogen, USA), 55 mM β-mercaptoethanol (Invitrogen, USA), 2 mM l-GlutaMAX (Invitrogen, USA), and 50 μg/ml penicillin/streptomycin (Millipore, USA) to form embryoid bodies (EBs). The medium was replaced every 2–3 days. On culture day 7, EBs were transferred to gelatin-coated plates and allowed to spontaneously differentiated for a further 2 weeks. The expression of smooth muscle actin (Abcam, USA), α-fetoprotein (Calbiochem, USA), and NESTIN (Millipore, USA), markers for three primitive germ layers, was determined by immunofluorescence.

Teratoma formation

Approximately 1 × 107 hESCs were suspended in 30% (v/v) Matrigel and transplanted intramuscularly into hind legs of 6–8-week-old nude mice. At 8–10 weeks post transplant, the fully formed teratomas were harvested and fixed with 4% (w/v) paraformaldehyde in PBS, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histological analysis.

Detection of α-thalassemia 1 SEA mutation

Detection of α-thalassemia 1 SEA mutation was performed as described previously by Winichagoon et al. [29]. Briefly, one or two blastomeres were removed from the embryos when they reached the eight-cell stage and transferred to a 0.2-ml reaction tube containing 2.5 μl lysis buffer and 50 μg/ml proteinase K (Invitrogen, USA). PCR was then performed to detect the mutation of α-globin genes. The primer sequences are presented in Additional file 1: Table S3.

Results

Derivation of hESCs by whole embryo culture and mechanical ICM dissection

To compare the efficiency of our MTP method with previously available hESC derivation methods, we firstly derived hESCs under feeder-based conditions using two standard hESC derivation methods, WEC and MID (Fig. 1a). For WEC, the zona pellucida (ZP) of three human blastocysts was removed by incubating the embryos with pronase enzyme. The zona-free embryos were then cultured on irradiated human foreskin fibroblasts (HFFs) in Nutristem medium, a commercial GMP-certified, xeno-free medium for hESC culture. After 3 days of culture, flattened outgrowths from the embryos were observed (Fig. 1b). However, most of those outgrowths mainly consisted of TE cells which disrupted the ICM organization. On culture day 5, no ICM outgrowth was observed and no hESC line was derived from those three embryos (Fig. 1b and Table 1).
Table 1

Efficiency of hESC derivation using WEC, MID, and MTP techniquesa

 

Number of embryosb

Outgrowth (day 3)

Number (%) of established hESC lines

WEC

3

3

0

MID

6

6

2 (33)

MTP

10

10

7 (70)

hESC human embryonic stem cell, WEC whole embryo culture, MID mechanical ICM dissection, ICM inner cell mass, MTP minimized trophoblast proliferation

aExperiments presented were performed by the same personnel

bAll embryos used were discarded embryos with genetic abnormalities, diagnosed by performing blastomere biopsy and preimplantation genetic diagnosis

For MID, ICM was mechanically isolated from the surrounding TE cells (Fig. 1c). Six ICM clumps were successfully isolated from six blastocysts (three with Hb Bart’s hydrops fetalis, two with trisomy 13, and one with trisomy 18). The isolated ICMs were then cultured on irradiated HFFs in Nutristem medium. After 3 days of culture, several distinct ICM organizations surrounded with a small number of TE cells were observed (Fig. 1d). Only two ICMs with Hb Bart’s hydrops fetalis can be further expanded to generate hESC lines, SiBart1 and SiBart2 (Additional file 2: Figure S1).

Decrease TE cell proliferation using a feeder-free culture system

It is possible that the feeder cells may promote TE cell proliferation which results in the low hESC derivation efficiency observed with the MID technique. We therefore cultured ICMs on plates coated with CELLstart or Matrigel in Nutristem media under hypoxic conditions without feeder cells. We observed that the ICMs attached to both the CELLstart and Matrigel-coated surfaces within the first day and the ICM clumps were prominent after 2 days of culture. Although TE cell proliferation and expansion was initially observed (Fig. 2a), those TE cells subsequently deteriorated on day 3 or 4. When the ICM clumps were picked and transferred onto fresh HFFs, they further expanded and were ready for passage on culture day 14 while the TE cells were no longer observed. This result indicates that culturing ICMs on CELLstart or Matrigel instead of HFFs can prevent TE cell proliferation and enhance ICM expansion (Fig. 2b).

Enhancement of hESC derivation using the MTP technique

Seven embryos (one parthenogenetic embryo and six aneuploid embryos including two with monosomy 13, two with trisomy 13, one with trisomy 18, and one with XYY syndrome; Fig. 3a) were studied. The ICMs from each embryo were isolated by MID and cultured on Matrigel-coated plates in Nutristem media under hypoxic conditions. On culture day 3, the ICM clumps were separated from the surrounding TE cells and transferred onto HFFs. At this stage, the ICM cells proliferated extensively and five hESC lines were successfully established with a derivation efficiency of 71.4%. Of these five hESC lines, two were derived from monosomy 13 embryos (Si1 and Si2) and one each from XYY embryo (Si3), trisomy 13 embryo (Si4), and parthenogenetic embryo (Si5).
Fig. 3
Fig. 3

Derivation of hESCs from aneuploid embryos using MTP under a feeder-free system. a Eight-cell-stage human embryos subjected to blastomere biopsy and their chromosomal abnormalities determined by fluorescence in-situ hybridization (FISH). b Mechanically isolated ICM (arrowheads) with large number of associated TE cells (arrows) detached from Matrigel-coated surface and degenerated after 4 days of culture. c Mechanically isolated ICM (arrowheads) carefully stripped of TE cells (arrows) remains attached to Matrigel-coated surface and continuously proliferated to form large hESC colonies while remaining TE cells degenerated. Scale bar: 50 μm

We also used the MTP method to derive hESCs from frozen–thawed embryos. ICMs were isolated from two frozen embryos with 3 pronuclei (3PN) by MID and cultured on Matrigel-coated plates in Nutristem media under hypoxic conditions. One hESC line (Si3PN) was then successfully established with a derivation efficiency of 50% (Additional file 3: Figure S2A). Karyotyping showed that Si3PN exhibited the 69, XXY karyotype (Additional file 3: Figure S2C). We also successfully used the MTP method to derive hESCs from an embryo with Hb Bart’s hydrops fetalis (SiAtha1) (Additional file 3: Figure S2B). Karyotyping reveals that SiAtha1 exhibited the normal 46, XY karyotype (Additional file 3: Figure S2D).

Taken together, the results show that our MTP method enhanced the hESC derivation efficiency from both normal and abnormal embryos. Using the MTP technique, seven hESC lines were successfully derived from 10 embryos with an overall efficiency of 70% (Table 2), the highest among other previous reports (Additional file 1: Table S1), and approximately 3-fold higher than the conventional methods, MID and WEC (Table 1, Additional file 1: Table S2). All hESC lines established in this study expressed typical pluripotent marker genes and proteins, and could differentiate to generate derivatives of all three embryonic germ layers both in vitro and in vivo (Additional file 4: Figure S3).
Table 2

hESC lines successfully derived by MTP technique

Type of embryo

Number of embryos

Number (%) of established hESC lines

Name of hESC line

Monosomy 13

2

2 (100)

Si1, Si2

Trisomy 13

2

1 (50)

Si4

47, XYY

1

1 (100)

Si3

Parthenogenetic

1

1 (100)

Si5

3PN (vitrified–thawed)

2

1 (50)

Si3PN

Alpha thalassemia

1

1 (100)

SiAtha1

hESC human embryonic stem cell, MTP minimized trophoblast proliferation

Derivation of hESCs in a feeder-free culture system using minimized TE cell contamination

To explore use of the MTP method for deriving hESCs under feeder-free conditions, we cultured the ICMs isolated by MID on Matrigel-coated plates throughout the entire culture period without transferring the cells onto HFFs. TE cells deteriorated after 3 days of culture while the ICM remained intact. However, when the culture was continued under this condition, the ICMs began to degenerate and detached from the culture surface on culture day 5 (Fig. 3b). When the detached ICMs were transferred onto HFFs for further expansion, they did not proliferate, resulting in the termination of culture.

Based on this observation, it is possible that an extensive deterioration of TE cells prior to the degeneration of ICMs might release some toxic molecules that exerted deleterious effects on ICM cells. To solve this problem, we tried to minimize the number of contaminated TE cells at the beginning of culture by carefully separating ICMs from the surrounding TE cells in the blastocysts using a fine pulled-glass needle. When those carefully isolated ICMs were cultured on Matrigel-coated plates, they attached to the culture surface and began to proliferate. Using this technique, the numbers of contaminated TE cells in culture were dramatically decreased and the ICM outgrowths proceed even after the remaining TE cells degenerated. After 8 days of culture, the ICM-derived colonies were mechanically dissociated and replated into fresh Matrigel-coated plates. The ICM-derived colonies which exhibited typical hESC morphology (Fig. 3c) could be expanded further and the hESC lines were successfully established. The results demonstrate that an optimized MTP method could be used to derive clinical-grade hESC lines under feeder-free and xeno-free medium (Fig. 4).
Fig. 4
Fig. 4

Derivation of clinical-grade hESCs by MTP under feeder-based and feeder-free conditions. To generate clinical-grade hESCs, mechanically isolated ICM initially cultured on GMP/clinical-grade extracellular matrix, such as rLaminin-521 or CELLstart, for a few days before being transferred onto GMP-grade HFFs (for feeder-based system) or fresh matrix-coated plates (for feeder-free system) to generate hESC colonies. ICM: inner cell mass, GMP: good manufacturing practice

Discussion

Previous reports using either the MID or WEC technique to derive hESCs indicated that TE cell outgrowth could suppress ICM expansion and cause them to degenerate, resulting in the low hESC derivation efficiency [3032]. In the present study, we successfully developed a more effective hESC derivation technique by minimizing the amount of contaminated TE cells during mechanical ICM dissection and using a feeder-free culture system to limit TE cell proliferation (MTP technique). The idea of using the MTP technique is to reduce TE cell proliferation and allow the ICM to expand during the first few days of culture. At this time, the ICM cells begin to acquire the embryonic stem cell phenotype as demonstrated by the upregulation of genes associated with self-renewal, such as Lin28a [33, 34]. It is of importance that the separation of ICM outgrowth from degenerated TE cells is critical for the successful derivation of hESCs under feeder-free conditions. Allowing degenerated TE cells to contact with ICM outgrowth results in ICM degeneration, possibly due to toxic substances or apoptosis signals released from the degenerating TE cells.

The hESC lines derived from various types of embryos using the MTP technique exhibited typical characteristics and functional properties of hESCs, and some lines (Si3PN and SiAtha1) preserved the original genetic mutations of the embryos that were used to derive them. The MTP technique can also be used to derive hESCs under xeno-free conditions, which is suitable for clinical-grade hESC derivation. We also showed that CELLstart, which is a nonanimal GMP-grade extracellular matrix, can be used to derive hESCs by our MTP technique.

In this study, we used human embryos with various chromosomal abnormalities to derive hESCs. Blastomere biopsy was performed when the embryos reached the eight-cell stage for PGD. Those embryos therefore consisted of approximately 12.5–25% fewer cells than intact embryos [35]. It is controversial whether blastomere biopsy affects embryonic development and the implantation rate. Previous studies demonstrated that blastomere biopsy did not affect blastocyst formation, embryonic implantation, and subsequent development of postimplantation embryos [3032, 3641]. However, there were reports suggesting that biopsied embryos showed delayed compaction, generated fewer hatched blastocysts, and had a lower implantation rate (39%) when compared with nonbiopsied embryos [4247]. Our results showed that blastomere biopsy did not affect the hESC derivation efficiency, indicating that the remaining number of blastomeres after biopsy is sufficient for generating ICM for subsequent hESC derivation by the MTP method.

Surprisingly, all hESC lines derived from four embryos with various chromosomal abnormalities exhibited a normal diploid karyotype (Additional file 5: Figure S4). However, this was not unprecedented as there was a report of deriving normal diploid hESCs from aneuploid embryos [48]. This may be due to a phenomenon called “mosaicism” found in small percentages of eight-cell-stage embryos at the time of PGD [49]. The meiotic nondisjunction occurring during germ cell formation and the mitotic nondisjunction occurring during the early cleavage stage of the embryos are mostly responsible for the mosaicism, resulting in affected embryos with both normal diploid cells and aneuploid cells [5052] (Additional file 6: Figure S5). It is therefore possible that our four abnormal embryos were affected by mosaicism and the normal diploid cells presented in those embryos might outcompete their aneuploid counterparts during the hESC derivation process, resulting in the generation of hESC lines with a normal karyotype (Si1, Si2, Si3, and Si4).

Conclusion

We report use of the MTP technique to derive hESCs from normal, disease, frozen, and parthenogenetic embryos with efficiency greater than that described previously. With this MTP technique, hESC lines can be successfully derived under feeder-free conditions in well-defined, xeno-free medium (Fig. 4). This methodology can be used to derive clinical-grade hESCs from a limited number of embryos for future therapeutic applications.

Abbreviations

cDNA: 

Complementary DNA

GMP: 

Good manufacturing practice

Hb: 

Hemoglobin

hESC: 

Human embryonic stem cell

HFF: 

Human foreskin fibroblast

ICM: 

Inner cell mass

iPSC: 

Induced pluripotent stem cell

IVF: 

In vitro fertilization

MID: 

Mechanical ICM dissection

MTP: 

Minimized trophoblast cell proliferation

Neu5Gc: 

N-Glycolylneuraminic acid

PBS: 

Phosphate buffer saline

PGD: 

Preimplantation genetic diagnosis

RT-PCR: 

Reverse transcription polymerase chain reaction

SEA: 

Southeast Asia

SIRB: 

Siriraj Institutional Review Board, Faculty of Medicine, Siriraj Hospital

TE: 

Trophoblast

WEC: 

Whole embryo culture

ZP: 

Zona pellucida

Declarations

Acknowledgements

The authors thank Prof. Peter Andrews, Prof. Harry Moore, from The University of Sheffield, Prof. Davor Solter, and Prof. Barbara Knowles for their valuable suggestion during hESC derivation. We would like to thank Dr Kamthorn Pruksananonda and Dr Ruttachuk Rungsiwiwut for kindly providing Chula2 hESCs.

Funding

This work was supported by The Thailand Research Fund (TRG5780180 to CLa and RTA488–0007 to SI) and the Commission on Higher Education (CHE-RES-RG-49 to SI). SI is a TRF Senior Research Scholar.

Availability of data and materials

All datasets in this article are included within the article and additional files.

Authors’ contributions

CLa contributed to conception and design, hESC derivation, the experiment, data analysis and interpretation, financial support, and manuscript writing. PC conducted the experiment and manuscript writing. RC provided blastocyst embryos in this study, and conducted data analysis and interpretation. SP, KS, CK, and OM provided blastocyst embryos in this study. PT contributed to histological analysis. SW contributed to cytogenetic analysis. CLo and PK contributed to data analysis and interpretation, and manuscript writing. YU and PS contributed to data analysis and interpretation. SI contributed to conception and design, financial support, and manuscript writing. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The study protocol was approved by Siriraj Institutional Review Board, Faculty of Medicine, Siriraj Hospital (SIRB), Mahidol University (Si338/2013). All experiments with human embryos were performed under the guidelines and regulations of SIRB, Mahidol University. The human embryos used in this study were obtained from the infertility unit, Siriraj Hospital. Informed consent was obtained from all couples that donated spare embryos following IVF treatment.

Competing interests

The authors declare no competing financial interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Siriraj Center of Excellence for Stem Cell Research (SiSCR), Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand
(2)
Division of Infertility and Reproductive Biology, Department of Obstetrics and Gynaecology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand
(3)
Department of Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand
(4)
Division of Medical Genetics, Department of Obstetrics and Gynaecology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand
(5)
Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand
(6)
Division of Cell Biology, Faculty of Medicine, Thammasat University, Pathumthani, 12120, Thailand

References

  1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science (New York, NY). 1998;282(5391):1145–7.View ArticleGoogle Scholar
  2. Chen AE, Egli D, Niakan K, Deng J, Akutsu H, Yamaki M, et al. Optimal timing of inner cell mass isolation increases the efficiency of human embryonic stem cell derivation and allows generation of sibling cell lines. Cell Stem Cell. 2009;4(2):103–6.View ArticlePubMedPubMed CentralGoogle Scholar
  3. Strom S, Holm F, Bergstrom R, Stromberg AM, Hovatta O. Derivation of 30 human embryonic stem cell lines—improving the quality. In Vitro Cell Dev Biol Anim. 2010;46(3–4):337–44.View ArticlePubMedPubMed CentralGoogle Scholar
  4. Solter D, Knowles BB. Immunosurgery of mouse blastocyst. Proc Natl Acad Sci U S A. 1975;72(12):5099–102.View ArticlePubMedPubMed CentralGoogle Scholar
  5. Stojkovic M, Lako M, Stojkovic P, Stewart R, Przyborski S, Armstrong L, et al. Derivation of human embryonic stem cells from day-8 blastocysts recovered after three-step in vitro culture. Stem Cells. 2004;22(5):790–7.View ArticlePubMedGoogle Scholar
  6. Klimanskaya I, Chung Y, Meisner L, Johnson J, West MD, Lanza R. Human embryonic stem cells derived without feeder cells. Lancet (London, England). 2005;365(9471):1636–41.View ArticleGoogle Scholar
  7. Tannenbaum SE, Turetsky TT, Singer O, Aizenman E, Kirshberg S, Ilouz N, et al. Derivation of xeno-free and GMP-grade human embryonic stem cells—platforms for future clinical applications. PLoS One. 2012;7(6):e35325.View ArticlePubMedPubMed CentralGoogle Scholar
  8. Strom S, Inzunza J, Grinnemo KH, Holmberg K, Matilainen E, Stromberg AM, et al. Mechanical isolation of the inner cell mass is effective in derivation of new human embryonic stem cell lines. Hum Reprod. 2007;22(12):3051–8.View ArticlePubMedGoogle Scholar
  9. Fan Y, Luo Y, Chen X, Sun X. A modified culture medium increases blastocyst formation and the efficiency of human embryonic stem cell derivation from poor-quality embryos. J Reprod Dev. 2010;56(5):533–9.View ArticlePubMedGoogle Scholar
  10. Wang Q, Fang ZF, Jin F, Lu Y, Gai H, Sheng HZ. Derivation and growing human embryonic stem cells on feeders derived from themselves. Stem Cells. 2005;23(9):1221–7.View ArticlePubMedGoogle Scholar
  11. Oh SK, Kim HS, Ahn HJ, Seol HW, Kim YY, Park YB, et al. Derivation and characterization of new human embryonic stem cell lines: SNUhES1, SNUhES2, and SNUhES3. Stem Cells. 2005;23(2):211–9.View ArticlePubMedGoogle Scholar
  12. Simon C, Escobedo C, Valbuena D, Genbacev O, Galan A, Krtolica A, et al. First derivation in Spain of human embryonic stem cell lines: use of long-term cryopreserved embryos and animal-free conditions. Fertil Steril. 2005;83(1):246–9.View ArticlePubMedGoogle Scholar
  13. Faden RR, Dawson L, Bateman-House AS, Agnew DM, Bok H, Brock DW, et al. Public stem cell banks: considerations of justice in stem cell research and therapy. Hast Cent Rep. 2003;33(6):13–27.View ArticleGoogle Scholar
  14. Crook JM, Peura TT, Kravets L, Bosman AG, Buzzard JJ, Horne R, et al. The generation of six clinical-grade human embryonic stem cell lines. Cell stem cell. 2007;1(5):490–4.Google Scholar
  15. Kim SJ, Lee JE, Park JH, Lee JB, Kim JM, Yoon BS, et al. Efficient derivation of new human embryonic stem cell lines. Mol Cells. 2005;19(1):46–53.PubMedGoogle Scholar
  16. Cowan CA, Klimanskaya I, McMahon J, Atienza J, Witmyer J, Zucker JP, et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med. 2004;350(13):1353–6.View ArticlePubMedGoogle Scholar
  17. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663–76.View ArticlePubMedGoogle Scholar
  18. Garitaonandia I, Amir H, Boscolo FS, Wambua GK, Schultheisz HL, Sabatini K, et al. Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions. PLoS One. 2015;10(2):e0118307.View ArticlePubMedPubMed CentralGoogle Scholar
  19. Na J, Baker D, Zhang J, Andrews PW, Barbaric I. Aneuploidy in pluripotent stem cells and implications for cancerous transformation. Protein Cell. 2014;5(8):569–79.View ArticlePubMedPubMed CentralGoogle Scholar
  20. Feng Q, Lu SJ, Klimanskaya I, Gomes I, Kim D, Chung Y, et al. Hemangioblastic derivatives from human induced pluripotent stem cells exhibit limited expansion and early senescence. Stem Cells. 2010;28(4):704–12.View ArticlePubMedGoogle Scholar
  21. Hu BY, Weick JP, Yu J, Ma LX, Zhang XQ, Thomson JA, et al. Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency. Proc Natl Acad Sci U S A. 2010;107(9):4335–40.View ArticlePubMedPubMed CentralGoogle Scholar
  22. Chin MH, Mason MJ, Xie W, Volinia S, Singer M, Peterson C, et al. Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression signatures. Cell Stem Cell. 2009;5(1):111–23.View ArticlePubMedPubMed CentralGoogle Scholar
  23. Kang E, Wang X, Tippner-Hedges R, Ma H, Folmes CD, Gutierrez NM, et al. Age-related accumulation of somatic mitochondrial DNA mutations in adult-derived human iPSCs. Cell Stem Cell. 2016;18(5):625–36.View ArticlePubMedGoogle Scholar
  24. Bouma MJ, van Iterson M, Janssen B, Mummery CL, Salvatori DCF, Freund C. Differentiation-defective human induced pluripotent stem cells reveal strengths and limitations of the teratoma assay and in vitro pluripotency assays. Stem Cell Rep. 2017;8(5):1340–53.View ArticleGoogle Scholar
  25. Simonson OE, Domogatskaya A, Volchkov P, Rodin S. The safety of human pluripotent stem cells in clinical treatment. Ann Med. 2015;47(5):370–80.View ArticlePubMedGoogle Scholar
  26. Nori S, Okada Y, Nishimura S, Sasaki T, Itakura G, Kobayashi Y, et al. Long-term safety issues of iPSC-based cell therapy in a spinal cord injury model: oncogenic transformation with epithelial-mesenchymal transition. Stem Cell Rep. 2015;4(3):360–73.View ArticleGoogle Scholar
  27. Tesarova L, Simara P, Stejskal S, Koutna I. The aberrant DNA methylation profile of human induced pluripotent stem cells is connected to the reprogramming process and is normalized during in vitro culture. PLoS One. 2016;11(6):e0157974.View ArticlePubMedPubMed CentralGoogle Scholar
  28. El Khatib MM, Ohmine S, Jacobus EJ, Tonne JM, Morsy SG, Holditch SJ, et al. Tumor-free transplantation of patient-derived induced pluripotent stem cell progeny for customized islet regeneration. Stem Cells Transl Med. 2016;5(5):694–702.View ArticlePubMedPubMed CentralGoogle Scholar
  29. Winichagoon P, Fucharoen S, Kanokpongsakdi S, Fukumaki Y. Detection of alpha-thalassemia-1 (Southeast Asian type) and its application for prenatal diagnosis. Clin Genet. 1995;47(6):318–20.View ArticlePubMedGoogle Scholar
  30. Monk M, Handyside AH. Sexing of preimplantation mouse embryos by measurement of X-linked gene dosage in a single blastomere. J Reprod Fertil. 1988;82(1):365–8.View ArticlePubMedGoogle Scholar
  31. Wilton LJ, Shaw JM, Trounson AO. Successful single-cell biopsy and cryopreservation of preimplantation mouse embryos. Fertil Steril. 1989;51(3):513–7.View ArticlePubMedGoogle Scholar
  32. Hardy K, Martin KL, Leese HJ, Winston RM, Handyside AH. Human preimplantation development in vitro is not adversely affected by biopsy at the 8-cell stage. Hum Reprod. 1990;5(6):708–14.View ArticlePubMedGoogle Scholar
  33. Tang F, Barbacioru C, Bao S, Lee C, Nordman E, Wang X, et al. Tracing the derivation of embryonic stem cells from the inner cell mass by single-cell RNA-Seq analysis. Cell Stem Cell. 2010;6(5):468–78.View ArticlePubMedPubMed CentralGoogle Scholar
  34. Nichols J, Smith A. The origin and identity of embryonic stem cells. Development. 2011;138(1):3–8.View ArticlePubMedGoogle Scholar
  35. McArthur SJ, Leigh D, Marshall JT, de Boer KA, Jansen RP. Pregnancies and live births after trophectoderm biopsy and preimplantation genetic testing of human blastocysts. Fertil Steril. 2005;84(6):1628–36.View ArticlePubMedGoogle Scholar
  36. Cohen J, Wells D, Munne S. Removal of 2 cells from cleavage stage embryos is likely to reduce the efficacy of chromosomal tests that are used to enhance implantation rates. Fertil Steril. 2007;87(3):496–503.View ArticlePubMedGoogle Scholar
  37. Cieslak-Janzen J, Tur-Kaspa I, Ilkevitch Y, Bernal A, Morris R, Verlinsky Y. Multiple micromanipulations for preimplantation genetic diagnosis do not affect embryo development to the blastocyst stage. Fertil Steril. 2006;85(6):1826–9.View ArticlePubMedGoogle Scholar
  38. Liu Y, Zhou C, Xu Y, Fang C, Zhang M. Pregnancy outcome in preimplantation genetic diagnosis cycle by blastomere biopsy is related to both quality and quantity of embryos on day 3. Fertil Steril. 2009;91(4 Suppl):1355–7.View ArticlePubMedGoogle Scholar
  39. Xu K, Montag M. New perspectives on embryo biopsy: not how, but when and why? Semin Reprod Med. 2012;30(4):259–66.View ArticlePubMedGoogle Scholar
  40. Sampino S, Zacchini F, Swiergiel AH, Modlinski AJ, Loi P, Ptak GE. Effects of blastomere biopsy on post-natal growth and behavior in mice. Hum Reprod. 2014;29(9):1875–83.View ArticlePubMedGoogle Scholar
  41. Ishii T. Reproductive medicine involving genome editing: clinical uncertainties and embryological needs. Reproductive biomedicine online. 2017;34:27–31. https://doi.org/10.1016/j.rbmo.2016.09.009.
  42. Kirkegaard K, Hindkjaer JJ, Ingerslev HJ. Human embryonic development after blastomere removal: a time-lapse analysis. Hum Reprod. 2012;27(1):97–105.View ArticlePubMedGoogle Scholar
  43. Bar-El L, Kalma Y, Malcov M, Schwartz T, Raviv S, Cohen T, et al. Blastomere biopsy for PGD delays embryo compaction and blastulation: a time-lapse microscopic analysis. J Assist Reprod Genet. 2016;33(11):1449–57.View ArticlePubMedPubMed CentralGoogle Scholar
  44. Staessen C, Verpoest W, Donoso P, Haentjens P, Van der Elst J, Liebaers I, et al. Preimplantation genetic screening does not improve delivery rate in women under the age of 36 following single-embryo transfer. Hum Reprod. 2008;23(12):2818–25.View ArticlePubMedGoogle Scholar
  45. Debrock S, Melotte C, Spiessens C, Peeraer K, Vanneste E, Meeuwis L, et al. Preimplantation genetic screening for aneuploidy of embryos after in vitro fertilization in women aged at least 35 years: a prospective randomized trial. Fertil Steril. 2010;93(2):364–73.View ArticlePubMedGoogle Scholar
  46. Scott RT Jr, Upham KM, Forman EJ, Zhao T, Treff NR. Cleavage-stage biopsy significantly impairs human embryonic implantation potential while blastocyst biopsy does not: a randomized and paired clinical trial. Fertil Steril. 2013;100(3):624–30.View ArticlePubMedGoogle Scholar
  47. Hardarson T, Hanson C, Lundin K, Hillensjo T, Nilsson L, Stevic J, et al. Preimplantation genetic screening in women of advanced maternal age caused a decrease in clinical pregnancy rate: a randomized controlled trial. Hum Reprod. 2008;23(12):2806–12.View ArticlePubMedGoogle Scholar
  48. Lavon N, Narwani K, Golan-Lev T, Buehler N, Hill D, Benvenisty N. Derivation of euploid human embryonic stem cells from aneuploid embryos. Stem Cells. 2008;26(7):1874–82.View ArticlePubMedGoogle Scholar
  49. Ravichandran K, Guzman L, Escudero T, Zheng X, Colls P, Jordan A, et al. Causes and estimated incidences of sex-chromosome misdiagnosis in preimplantation genetic diagnosis of aneuploidy. Reprod BioMed Online. 2016;33(5):550–9.View ArticlePubMedGoogle Scholar
  50. Vanneste E, Voet T, Le Caignec C, Ampe M, Konings P, Melotte C, et al. Chromosome instability is common in human cleavage-stage embryos. Nat Med. 2009;15(5):577–83.View ArticlePubMedGoogle Scholar
  51. Biancotti JC, Narwani K, Buehler N, Mandefro B, Golan-Lev T, Yanuka O, et al. Human embryonic stem cells as models for aneuploid chromosomal syndromes. Stem Cells. 2010;28(9):1530–40.View ArticlePubMedGoogle Scholar
  52. Yuan L, Liu JG, Hoja MR, Wilbertz J, Nordqvist K, Hoog C. Female germ cell aneuploidy and embryo death in mice lacking the meiosis-specific protein SCP3. Science (New York, NY). 2002;296(5570):1115–8.View ArticleGoogle Scholar

Copyright

© The Author(s). 2018

Advertisement