All Things Stem Cell

One of the major hurdles that needs to be overcome in the field of regenerative medicine is the issue of immune rejection, or preventing a patient’s body from
rejecting a tissue transplant from a foreign donor. Consequently, researchers have increasingly focused on ways to regenerate damaged or diseased tissues in a patient by using the patient’s own tissues, which should not trigger an immune response. At this point in time, there are primarily two types of stem cells that hold the greatest promise for use in regenerative medicine where immune rejection is a significant concern: human induced pluripotent stem cells (iPSCs) and cells made through a process called somatic cell nuclear transfer (SCNT). This article will focus on recent SCNT improvements, but we’ll re-visit iPSCs briefly for comparison’s sake.

Applying Somatic Cell Nuclear Transfer in the Creation of Dolly the Cloned Sheep. Dolly the sheep was cloned through somatic cell nuclear transfer (SCNT). An adult cell from the mammary gland of a Finn-Dorset ewe acted as the nuclear donor; it was fused with an enucleated egg from a Scottish Blackface ewe, which acted as the cytoplasmic (or egg) donor. An electrical pulse acted to fuse the cells and activate the oocyte after injection into the surrogate mother ewe. A successfully implanted oocyte developed into the lamb Dolly, a clone of the nuclear donor, the Finn-Dorset ewe. SCNT may also be used to create patient-specific stem cells with great therapeutic potential.

Human induced pluripotent stem cells: The history and biology of human iPSCs were explored previously in “Induced Pluripotent Stem Cells: A New Stem Cell Line with a Long History.” In essence, iPSCs, which were first created with mouse cells in 2006 (Takahashi and Yamanaka, 2006) and then with human cells in 2007 (Yu et al., 2007; Takahashi et al., 2007), are adult cells that have been “reprogrammed” to an embryonic stem cell (ESC) state. This reprogramming is done by forcing adult cells to express proteins that are essential to the ESC identity (by transducing the adult cells with a retrovirus vector that contains the DNA for the key proteins). Consequently, human iPSCs look and behave nearly indistinguishably from hESCs. Like hESCs, iPSCs are pluripotent (they can become any cell type) and proliferate virtually indefinitely, both features which are important for use in regenerative medicine.

However, while great improvements have been made to make this technology closer to the clinic (such as multiple approaches to create iPSCs that do not have the reprogramming genes randomly integrated into their genomes [Yu et al., 2009; Zhou et al., 2009]), and it may someday be used to generate patient-specific ESC-like cells, the technology is not quite there yet. (Other similar technologies, such as “direct reprogramming,” are also being explored for the generation of patient-specific cells, but, again, this approach also has a ways to go.)

Somatic cell nuclear transfer: SCNT technology significantly predates iPSCs, and in many ways formed the basis for the idea of iPSCs. In SCNT, the nucleus from a somatic cell (an adult cell that is not a sperm or egg, i.e. not the gametes) is implanted into an egg, which already had its own nucleus removed. The egg amazingly reprograms the nucleus to become embryonic again; it’s been found that SCNT causes some 10,000 to 12,000 genes to be expressed (turned into protein) that are normally associated only with embryonic development (Niemann et al., 2008). The newly formed embryo (technically called a blastocyst) can then be implanted into a surrogate mother, and potentially become an adult organism. The organism is a clone of the animal that donated the nucleus. Although nuclear transfer studies have been conducted since the late 1930s (primarily in amphibians using nuclei donated from embryos, not adult tissues) (Spemann, 1938), it wasn’t until 1997 that the first widely-accepted successful use of SCNT was reported: Dolly the sheep was born, and she was the first cloned animal from an adult cell, and the first cloned mammal (Wilmut et al., 1997). Since Dolly, several other animals have been successfully cloned, though many problems still remain (the frequency of successful development is relatively low, as SCNT-derived embryos usually result in about 0 to 10% live births) (Wilmut et al., 1997; Wakayama et al., 1998; Solter, 1998; McKinnell and Di Bernardino, 1999; Gurdon and Byrne, 2003, Beyhan and Cibelli, 2008).

Therapeutic cloning: While SCNT has been long-explored for its ability to create cloned animals like Dolly and others (a practice called “reproductive cloning”), SCNT has other very appealing applications that do not involve the creation of an entire animal, such as “therapeutic cloning.” The goal of therapeutic cloning is to use SCNT technology to create patient-specific embryonic stem cells for medical therapies. These much sought-after cells are being labeled nuclear transfer stem cells (NTSCs), but they are essentially ESCs. While using SCNT to clone an entire animal has been fraught with developmental challenges, using SCNT to create NTSCs may be less difficult because it only requires a very early stage embryo, a blastocyst, to be formed (and NTSCs from a blastocyst may be less compromised by developmental abnormalities than an entire animal would be) (Beyhan and Cibelli, 2008). NTSCs have now been created from many different model animals.

SCNT in the Mouse: In 2000, the first embryonic stem cell line was generated from mice using SCNT (Munsie et al., 2000). After SCNT was performed, when the embryos reached the blastocyst stage the ESCs were isolated as usual. These SCNT-derived mouse ESCs (or NTSCs) were reported to be pluripotent and behave similarly to normally-derived mouse ESCs. Not only could NTSCs be successfully derived in this way, but multiple studies in mice have shown that SCNT can also be used to repair genetic diseases. This has been done by taking the nucleus from an adult cell of a mouse with a genetic defect, performing SCNT on the nucleus to create ESC lines, fixing the defect in these cell lines (using standard genetics techniques), and then transplanting the repaired (and differentiated) cells back into the original donor mouse (for descriptions of these studies, see Saric et al., 2009). The mice are virtually cured of their genetic diseases this way

SCNT in Non-Human Primates: In 2007, two ESC (or NTSC) lines were created from a rhesus macaque using SCNT (Byrne et al., 2007). The researchers took nuclei from the skin fibroblasts of a female rhesus macaque and had these undergo SCNT in the eggs of another female macaque. Although these cells could differentiate properly and express pluripotent markers, the generation efficiency was low; 304 eggs were used to create just these two ESC lines. (This was thought to be due to incomplete reprogramming of the donor nucleus.) However, two years later the same group reported a three-fold increase in NTSC generation from rhesus macaques using improved techniques, reportedly creating NTSC lines at levels similar to those seen with the generation of ESC lines from normally fertilized embryos (Sparman et al., 2009). These NTSCs also displayed proper epigenetic reprogramming, similar to normal ES cells, which had been of concern (Sparman et al., 2009).

Humans and SCNT: Currently, a scarcity of donated human eggs is probably the largest hurdle to creating patient-specific, human ESCs through SCNT. Not only are more eggs needed, but these eggs need to have certain qualities to work most efficiently in SCNT (they must be young, high-quality, and at metaphase II stage) (Cervera and Stojkovic, 2008). There are also many technical issues that need to be optimized (such as removing the least amount of cytoplasm from the egg when enucleating it, and avoiding the use of certain nuclear stains while doing this, though reports vary on even the importance of these kinds of aspects). In 2004, Hwang and colleagues reported having successfully created hESCs from a SCNT blastocyst for the first time, but their work was proven to be fraudulent. Because of these falsified studies, subsequent researchers have had to go to great lengths to show that their SCNT data is valid, making it a more difficult field to publish in.

Because of the lack of human eggs for creating human blastocysts from SCNT, researchers have pursued using eggs donated from other animals (but nuclei from adult human cells) for SCNT. This technique is called human-animal interspecies SCNT (iSCNT). Many ethical concerns have been raised over the use of this technique, especially since an iSCNT embryo contains a human nucleus, but animal mitochondrial DNA (as well as traces of human mitochondrial DNA from the nuclear transfer). ( For more on the ethical implications of these practices, see Skene et al., 2009.)

Some reports have suggested that iSCNT embryos are not “healthy,” primarily because the donated nucleus often does not become fully reprogrammed to an embryonic state. In one such study, a group used chimpanzee somatic cell nuclei with cow eggs, but the embryos did not develop past the 8- to 16-cell stages. While epigenetic changes took place that appeared normal, it turned out that many developmentally important genes were not actually being expressed at all (Nisker et al., 2010). There have also been reports of iSCNT being performed using human and animal cells, such as with rabbit eggs and human nuclei (Chen et al., 2003), though there are still many ethical and developmental concerns surrounding iSCNT.

Despite the lack of available, donated human eggs and fraudulent work of Hwang and colleagues, several stem cell-oriented companies collaboratively published in 2008 the successful creation of human blastocysts from SCNT (French et al., 2008). In this study, 21 human eggs were obtained and each was combined with a nucleus from an adult, somatic (specifically fibroblast) cell through SCNT. From the 21 eggs and nuclei, five blastocysts were created. However, only one was rigorously and conclusively shown to have genomic and mitochondrial DNA from the nucleus donor (though two others also had partial supporting data), suggesting only one true clone was created.

The Future of SCNT: Although much work remains to be done before SCNT is a viable strategy for the production of patient-specific ESCs, there is enormous potential it could fulfill when it does reach that point. Not only may a patient have cells made that are specific to them and available as virtually any cell type they should need (such as insulin-producing beta cells for a patient with diabetes), but, for example, if they have a genetic disease this too may be cured (as was demonstrated in mice using this technology). Researchers could also create cell lines from patients to study specific diseases in the laboratory, greatly advancing our understanding of these conditions (though this is currently being explored with iPSCs). Lastly, as our ability to create ESC lines from different stage embryos and from fewer cells in these embryos, it should become easier to create NTSCs from SCNT-generated embryos (Cervera and Stojkovic, 2008). As SCNT research has come close to being completely banned in the United States, it’s important not only for researchers to take proper ethical precautions, but for everyone to keep in mind the great potential this technology holds.

References:

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