Hum. Reprod. Advance Access originally published online on July 8, 2005
Human Reproduction 2005 20(11):2987-2989; doi:10.1093/humrep/dei197
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
OPINION |
Human embryonic stem cell lines are contaminated: what should we do?
1 In Vitro Fertilization Laboratory, Tomball Regional Hospital, TX 77375, USA and 2 Institute of Obstetrics and Gyncology, Guangzhou Second People’s Hospital, Guangzhou, China
3 To whom correspondence should be addressed. E-mail: wangweihua11{at}yahoo.com
 |
Abstract |
|---|
 Top Abstract IntroductionSafe sources of oocytes...IVF techniques avoiding use...Isolation of ICM with...Culture of hES cell...Derivation of hES cells...References |
|---|
Human embryonic stem (hES) cells have the potential to differentiate into any desired cells and to be used in cell replacement therapies for some diseases. However, existing hES cell lines would not be suitable for the therapies as they are contaminated with other biological products. In order to produce the safest hES cell lines for therapeutic purposes, all steps for the establishment of hES cell lines must avoid the use of animal serum and/or animal feeder cell lines. Safe and fast approaches to producing hES cell lines based on recent research advances in both humans and animals have been suggested.
Key words: contamination/embryonic stem cells/human/IVF/safety
|
Introduction |
|---|
|
TopAbstractIntroductionSafe sources of oocytes...IVF techniques avoiding use...Isolation of ICM with...Culture of hES cell...Derivation of hES cells...References |
|---|
Under appropriate culture conditions, the inner cell mass (ICM) isolated from blastocysts can form embryonic stem (ES) cells. ES cells can be maintained in an undifferentiated state in culture, retaining the potential to differentiate into all types of cells. In humans, it is predicted that ES cells can be used to create specialized cells using appropriate culture conditions that enable undifferentiated cells to become differentiated. These cells can be used to treat a wide range of diseases, such as diabetes and Parkinson’s disease (Thomson et al., 1998
). Human ES (hES) cell research has provoked considerable debate: many scientists have questioned whether the existing cell lines would be adequate for the therapies as they are contaminated with other biological products. Indeed, recently, researchers identified an animal molecule, Neu5Gc, that has contaminated one of the stem cell lines in the National Institutes of Health stem cell registry (Martin et al., 2005). The contamination is believed also to have occurred in other existing cell lines because animal feeder layer and serum were used during the cell line production (from IVF to ES cell culture). Therefore, it is possible that the tissues developed by using contaminated stem cells may result in a deleterious immune reaction and/or rejection of the transplanted cells (Martin et al., 2005). As suggested by Martin et al. (2005), it would be safer to start over again with newly derived hES cells that have never been exposed to any animal products.
Currently, hES cell lines are being maintained or established mainly by using existing hES cell lines (Amit et al., 2004) or donated embryos (Klimanskaya et al., 2005) from human infertility clinics. However, embryos from infertility clinics may also become contaminated, especially by some human pathogens, such as human immunodeficiency virus (HIV), hepatitis B and C viruses, and other infectious agents. All infertility clinics may not screen infertile patients for infections; this may have an influence on the safety of hES cell lines if they will be used for transplantation. In addition, serum products from various sources have been used in media during IVF and early embryo culture, thus contamination is still possible although human serum supplements used in IVF have been screened for these pathogens.
Fetal bovine serum (FBS) has been used in the culture of feeder cells (Thomson et al., 1998), which are believed to support hES cell growth. However, as mentioned above, animal molecules from FBS may contaminate the feeder cells which in turn may contaminate hES cell lines. There are also concerns that viruses and other pathogens could be transmitted from feeder cells and FBS to patients, thus every system for the culture of hES cells should avoid using animal feeder cells and FBS. Although it may be safe to use human serum after careful screening of serum donors (Bongso and Richards, 2004), unknown pathogens may still exist in donor serum. Experience with organ and tissue transplantation has shown that many infectious agents can be transmitted from human donor cells to recipients. Therefore, in order to produce the safest hES cell lines for therapeutic purposes, all steps for the establishment of hES cell lines must be safe and hygienic. The following steps provide a safe direction and a fast approach to produce hES cell lines based on recent research advances in both humans and animals.
|
Safe sources of oocytes and embryos |
|---|
|
TopAbstractIntroductionSafe sources of oocytes...IVF techniques avoiding use...Isolation of ICM with...Culture of hES cell...Derivation of hES cells...References |
|---|
As mentioned above, most existing hES cell lines have been grown in animal-derived materials that pose a risk of contamination. New cell lines produced using donated frozen human embryos from human infertility clinics still have risks of contamination with infectious viruses. Even though the patients are screened for these viruses, there may be a risk of contamination in the embryo storage tank through storage of both non-infectious and infectious embryos in the same tank. Therefore, embryos donated from infertility clinics should be carefully chosen for production of hES cell lines, at least for future therapeutic purposes. For example, both male and female partners must undergo HIV and hepatitis B and C virus screening prior to infertility treatment. Alternative approaches to obtaining embryos include directly using donated oocytes and/or sperm. This has been done when hES cell lines were derived from cloned human embryos (Hwang et al., 2004
, 2005). These donors must be carefully screened for any possible infectious diseases and perhaps even genetic diseases. |
IVF techniques avoiding use of serum |
|---|
|
TopAbstractIntroductionSafe sources of oocytes...IVF techniques avoiding use...Isolation of ICM with...Culture of hES cell...Derivation of hES cells...References |
|---|
Currently, the methods used in human IVF laboratories for oocyte culture, IVF and early embryo development (to blastocyst stage) all involve media which are supplemented with proteins: human serum, synthetic serum substitute or human serum albumin. Again, all these proteins are derived from human sources and any contamination from one of the serum donors would contaminate the entire batch. Thus, they are still not safe for long-term consideration. Recently, a recombinant human albumin has been produced and supplementation of this protein provides the same, perhaps even better, results in terms of fertilization, early development and implantation (Blake et al., 2002
; Bungum et al., 2002). Therefore, recombinant serum albumin should be used for IVF procedures leading to hES cell production. |
Isolation of ICM with chemical or mechanical methods |
|---|
|
TopAbstractIntroductionSafe sources of oocytes...IVF techniques avoiding use...Isolation of ICM with...Culture of hES cell...Derivation of hES cells...References |
|---|
Isolation of the ICM is a very important step in the culture of ES cells. ICM are usually isolated from blastocysts by immunosurgery. However, antiserum used in immunosurgery is produced from certain experimental animals. Again, contamination in the antiserum may limit its clinical application. Therefore, a technology which avoids the use of animal products is necessary. Recently, an enzyme digestion method was used to isolate ICM from porcine blastocysts (Li et al., 2003
). This method uses 0.25% trypsin–0.04% EDTA solution to digest trophoblasts of the embryos, and with the aid of two fine needles and a pulled mouth micropipette, intact and healthy ICM are easily isolated from blastocysts. It has been found that the enzyme digestive method is much simpler than immunosurgery (Li et al., 2004). Because mouth pipetting may cause embryo-to-manipulator viral transmission (Steyaert et al., 2000) and/or manipulator-to-embryo transmission, alternative methods should be used, such as hand-manipulated devices. Other methods, such as a laser drill, that mechanically isolate the ICM from blastocysts should be considered. In other words, we should avoid using immunosurgery to isolate ICM for establishment of hES cell lines. |
Culture of hES cell line without serum and feeder cells |
|---|
|
TopAbstractIntroductionSafe sources of oocytes...IVF techniques avoiding use...Isolation of ICM with...Culture of hES cell...Derivation of hES cells...References |
|---|
Feeder layer cells can secret certain types of cytokines, such as leukaemia inhibitory factor (LIF), which may stimulate ES cell growth and inhibit their differentiation. Serum contains various growth factors that are beneficial to ES cell growth. This is the reason that almost all ES cell lines are produced and maintained in conditions containing both feeder layer and serum. By using a serum replacement that contains transforming growth factor
1, LIF, basic fibroblast growth factor and a fibronectin matrix, Amit et al. (2004) successfully maintained hES cell lines in a serum- and feeder-layer-free condition. Recently, Klimanskaya et al. (2005) used extracellular-matrix-coated plates to maintain six existing hES cell lines and then established one new hES cell line without serum and feeder cell layer. This is a significant advance in hES cell culture. However, the authors used existing hES cell lines (Amit et al., 2004; Klimanskaya et al., 2005) or used immunosurgery to isolate ICM (Klimanskaya et al., 2005) in their studies; therefore, existing hES cell lines may have already been exposed to potential contamination and ICM isolated by immunosurgery may still have animal products in the derived cell line. |
Derivation of hES cells from embryos produced by therapeutic cloning |
|---|
|
TopAbstractIntroductionSafe sources of oocytes...IVF techniques avoiding use...Isolation of ICM with...Culture of hES cell...Derivation of hES cells...References |
|---|
Although some countries allow the creation of human embryos or the use of supernumerary embryos produced in infertility clinics for research, such as derivation of hES cell lines (Siegel, 2004
), we are still concerned about the ethics of using these embryos. Many people hold strong views on the moral status of the human embryo (Taver, 2004). Very few patients are willing to donate their embryos for research (Barratt et al., 2004). Furthermore, donated supernumerary embryos from infertility clinics are of suboptimum quality and the genetic stability of the derived cell lines is still unknown (Barratt et al., 2004). We all know that there is great potential for hES cells in therapeutic applications, where the derived cells will be transplanted into patients. Therefore, two issues, safety and immunocompatibility, are of considerable significance. One approach is to produce safe hES cells as mentioned above. The other approach is to derive hES cells directly from oocytes through therapeutic cloning. Recently, patient-specific immunomatched hES cell lines have been established by therapeutic cloning technology (Hwang et al., 2005). If the conditions could provide for the differentiation of hES cells to desired cells, clinical applications of these cells would become possible. As nuclear transfer and ES cell culture techniques are becoming more efficient, new cell lines could be quickly established (Hwang et al., 2005); thus, rather than using human embryos, the utilization of oocytes (Brison and Lieberman, 2003) coupled with nuclear transfer (Hwang et al., 2004, 2005) to establish hES cell lines would become more attractive in the near future. In summary, combined technology should be used to produce new and safer hES cell lines so that the cells derived from these cell lines can be used in human therapies. All steps during ES cell line production should be screened for safety.
|
References |
|---|
|
TopAbstractIntroductionSafe sources of oocytes...IVF techniques avoiding use...Isolation of ICM with...Culture of hES cell...Derivation of hES cells...References |
|---|
Amit M, Shariki C, Margulets V and Itskovitz-Eldor J (2004) Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod 70,837–845.
Barratt CLR, St John JC and Afnan M (2004) Clinical challenges in providing embryos for stem-cell initiatives. Lancet 364,115–118.[CrossRef][Web of Science][Medline]
Blake D, Svalander P, Jin M et al (2002) Protein supplementation of human IVF culture media. J Assist Reprod Genet 19,137–143.[CrossRef][Web of Science][Medline]
Bongso A and Richards M (2004) History and perspective of stem cell research. Best Pract Res Clin Obstet Gynaecol, 18, 827–842.
Brison DR and Lieberman BA (2003) Use eggs, not embryos, to derive stem cells. Br Med J 327,872.
Bungum M, Humaidan P and Bungum L (2002) Recombinant human albumin as protein source in culture media used for IVF: a prospective randomized study. Reprod Biomed Online 4,233–236.[Medline]
Klimanskaya I, Chung Y, Meisner L et al (2005) Human embryonic stem cells derived without feeder cells. Lancet 365,1636–1641.[CrossRef][Web of Science][Medline]
Hwang WS, Ryu YJ, Park JH et al (2004) Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. Science 303,1669–1674.
Hwang WS, Roh SI, Lee BC et al (2005) Patient-specific embryonic stem cells derived from human SCNT blastocysts. Science 308,1777–1783.
Li M, Zhang D, Hou Y et al (2003) Isolation and culture of embryonic stem cells from porcine blastocysts. Mol Reprod Dev 65,429–434.[CrossRef][Medline]
Li M, Zhang D, Hou Y et al (2004) Improved isolation and culture of embryonic stem cells from Chinese miniature pig. J Reprod Dev 50,237–244.[Medline]
Martin MJ, Muotri A, Gage F and Varki A (2005) Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat Med 11,228–232.[CrossRef][Web of Science][Medline]
Siegel AW (2004) Temporal restrictions and the impasse on human embryonic stem-cell research. Lancet 364,215–218.[Medline]
Steyaert SR, Leroux-Roels GG and Dhont M (2000) Infections in IVF: review and guidelines. Hum Reprod Update 6,432–641.
Taver CA (2004) International policy failures: cloning and stem-cell research. Lancet 364,209–214.[Medline]
Thomson JA, Joseph IE, Shapiro SS et al (1998) Embryonic stem cell lines derived from human blastocysts. Science 282,1145–1147
Submitted on April 16, 2005; resubmitted on June 12, 2005; accepted on June 16, 2005.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||