Fertilization and early embryology: Isolation and culture of inner cell mass cells from human blastocysts

Bongso, Ariff; Fong, Chui‐Yee; Ng, Susanna S.S.; Ratnam, S. S.

doi:10.1093/oxfordjournals.humrep.a138401

Cited by 243 publications

(127 citation statements)

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“…This procedure, however, runs a much greater risk of trophectoderm overgrowth than the other two methods because the entire trophectoderm is cultured along with the ICM. These trophectodermal cells often hinder the growth of the ICM during the culture period and render the establishment of hESC lines rather problematic [18,19]. It was recently reported that several ESC lines were derived from cloned human embryos using whole-embryo culture method [11].…”

Section: Whole-embryo Culture Methodsmentioning

confidence: 99%

Methods for Derivation of Human Embryonic Stem Cells

Kim

Park

et al. 2005

STEM CELLS

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The expanded blastocysts, developed from 2PN-stage embryos, are generally divided into three categories: a good blastocyst containing a large and distinguishable inner cell mass (ICM), a blastocyst with a small and distinct ICM, and a blastocyst with a poorly defined ICM. In this study, we introduce methods for the derivation of human embryonic stem cells (hESCs) depending on the quality of the blastocysts. An immunosurgical method was used for the good expanded blastocysts. This method, however, raises the probability of ICM loss in cases of hESC derivation from blastocysts with smaller or indistinct ICMs. Furthermore, this method is also associated with a risk of the contamination of the hESCs with animal pathogens. To overcome these shortcomings, the partial-or whole-embryo culture method was used. For blastocysts with no visible ICM, the whole-embryo culture method was used to establish hESCs via the seeding of the entire blastocyst without its zona pellucida directly on a STO feeder layer. However, trophectodermal overgrowth tends to hinder the expansion of the ICM during the initial steps of hESC derivation. Therefore, the partial-embryo culture method was developed to establish hESCs from blastocysts with smaller ICMs. The surgical isolation of the region containing the ICM with an ultra-fine glass pipette alleviates trophectoderm overgrowth. This method is also applicable to blastocysts with large and distinct ICMs, and the efficiency of this method is comparable to that of the immunosurgical method.

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Section: Whole-embryo Culture Methodsmentioning

confidence: 99%

Methods for Derivation of Human Embryonic Stem Cells

Kim

Park

et al. 2005

STEM CELLS

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“…One of the mysterious properties of hESCs is that they are ''social'' cells that remain undifferentiated for long periods of time if propagated in clusters and not as single cells [Bongso et al, 1994[Bongso et al, , 2005. In all the conventional teratoma assays using SCID mice, teratomas are produced after injection of clusters of hESCs.…”

Section: Approaches To Eliminate Rogue Undifferentiated Hescs and Tummentioning

confidence: 99%

Taking stem cells to the clinic: Major challenges

Bongso

Fong

Gauthaman

2008

J of Cellular Biochemistry

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162

111

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Stem cell therapy offers tremendous promise in the treatment of many incurable diseases. A variety of stem cell types are being studied but human embryonic stem cells (hESCs) appear to be the most versatile as they are pluripotent and can theoretically differentiate into all the tissues of the human body via the three primordial germ layers and the male and female germ lines. Currently, hESCs have been successfully converted in vitro into functional insulin secreting islets, cardiomyocytes, and neuronal cells and transfer of such cells into diabetic, ischaemic, and parkinsonian animal models respectively have shown successful engraftment. However, hESC-derived tissue application in the human is fraught with the problems of ethics, immunorejection, tumorigenesis from rogue undifferentiated hESCs, and inadequate cell numbers because of long population doubling times in hESCs. Human mesenchymal stem cells (hMSC) though not tumorigenic, also have their limitations of multipotency, immunorejection, and are currently confined to autologous transplantation with the genuine benefits in allogeneic settings not conclusively shown in large controlled human trials. Human Wharton's jelly stem cells (WJSC) from the umbilical cord matrix which are of epiblast origin and containing both hESC and hMSC markers appear to be less troublesome in not being an ethically controversial source, widely multipotent, not tumorigenic, maintain ''stemness'' for several serial passages and because of short population doubling time can be scaled up in large numbers. This report describes in detail the hurdles all these stem cell types have to overcome before stem cell-based therapy becomes a genuine reality.

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“…Taking into account that we had used the laser drill for the good-quality blastocysts and that the concealment of the ICM by the trophectoderm cells is the only disadvantage of the whole-embryo culture method [6], we must continue to improve the laser-drill technique so that the trophectoderm cells are destroyed and do not interfere with ICM adhesion [12]. Curiously, the blastocysts treated with the laser at day + 1 formed a pseudotrophectoderm, with a few cells not destroyed by the laser because they were near the ICM ( Figure 3E).…”

Section: Discussionmentioning

confidence: 99%

“…The ICM could usually be separated and isolated from the trophectoderm using immunosurgery [5], with mechanical processes [6], with whole-embryo culture of the blastocysts and partial-embryo culture methods [7] or single blastomeres [8].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of a laser technique to isolate the inner cell mass of murine blastocysts

Cortés¹,

Cobo²,

Catalina³

et al. 2007

Biotech and App Biochem

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hESCs (human embryonic stem cells) are pluripotent cells derived from the ICM (inner cell mass) of blastocysts that can be used to derive several kinds of cells of the human body for the treatment of some previously untreated diseases. In considering the future use of hESCs in regenerative medicine and cell-therapy programmes, several research centres have begun projects involving the derivation of hESC lines using spare human embryos from IVF (in vitro fertilization) cycles. In some stem-cell banks, such as ours, the law also permits us to obtain these cell lines. The low availability of spare IVF human embryos, and the low rate of success in the derivation of hESC lines, give these embryos a great research value that limits experiments with new techniques. The use of murine embryos would be a good model with which to do research to discover the best methodologies to use in order to derive new hESC lines. The aim of the present study was to evaluate a new method of isolation of the ICM and derivation of ESC lines in a murine blastocyst model using laser drilling to eliminate the trophectoderm cells and compare it with the usual control method consisting of culturing the whole murine blastocyst. We also tested the adhesion and growth of primary colonies of mESCs (murine ESCs) over two different growth surfaces, namely an MEF (inactive murine fibroblastic feeder layer) or gelatin-coated dishes, in order to achieve the best culture conditions for future derivation of human stem-cell lines for application in human transplantation.

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Fertilization and early embryology: Isolation and culture of inner cell mass cells from human blastocysts

Cited by 243 publications

References 0 publications

Methods for Derivation of Human Embryonic Stem Cells

Methods for Derivation of Human Embryonic Stem Cells

Taking stem cells to the clinic: Major challenges

Evaluation of a laser technique to isolate the inner cell mass of murine blastocysts

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