Stem cells, their use as a cell-based therapy

Organ transplants are now a routine procedure. However, with the shortage of organ donors and potential immunological rejection, these are the two major challenges leading to transplant failure. Several possible solutions to both these problems are being tried and tested. Autologous grafts, involving the harvesting of tissue from one part of the body to repair another are often performed to avoid immunological rejections. Another possible solution to avoid potential immunological rejections and that also eliminates the necessity of employment of whole organs and/or tissue, is the use of special cells called stem cells. As stem cells are undeveloped (non-specialized) cells, they possess the ability to become any type of cell and so form any type of tissue including bone, muscle, nerve etc. This is because of their self-renewal and pluripotency characteristics which renders them with the potential to advance current therapies in tissue regeneration and/or engineering. Hence, with stem cell therapy, the idea is to somehow isolate such cells, multiply and process them in vitro to eventually utilize them in the replacement of damaged tissue. Many disease conditions could then be treated in this manner.

There are many different types of stem cells and these are present at all stages of an organism’s life from the early embryo to adult stages. Nonetheless the ideal option in facilitating stem cell based therapy would be through the isolation of embryonic stem from a patient, which might be difficult for treatment of adult patients. But technologies such as the nuclear transfer (cloning) technique have provided a platform for reversing the normal direction of cell differentiation; resulting in the reprogramming of the nucleus of an adult cell, thus allow suitable stem cells to be engineered from an adult cell. The two potential methods for obtaining suitable stem cells for cell-based therapy which are also ideal for patient-specific purposes are illustrated in figure 6.1.

Figure 6.1: In (A) on the left side of the figure, stem cells are obtained for therapy via redirecting one type of adult stem cells into another i.e. blood to nerve stem cells in this example. In (B) on the right side, an adult cell is reprogrammed into an early embryo cell to generate the necessary stem cells (online access date – 7. 1.10 (1)).

One of these methods involves the isolation and subsequent reprogramming of one kind of stem cells into another type of stem cell. For instance, in figure 6.1 (A) is depicted blood stem cells converted into nerve stem cells, which in turn could be used to cure various kinds of nerve cell disorders. On the contrary, cell nuclear replacement technology is where a biopsy is collected from a patient and by nuclear transfer, reprogramming of an adult cell into an early embryo is achieved (figure 6.1 (B)). The latter is achieved through removing the oocyte’s (unfertilized egg) own nucleus and replacing it with that of an adult donor cell’s nucleus resulting in the artificial creation of an embryo. The availability of the embryonic stem cells would then be through an in vitro culturing of the blastocyst stage and the later harvesting of its inner cell mass. Embryonic stem cell differentiation performed using a host of various techniques would then permit the formation of various cell types relevant for cell based therapies that are also patient-

specific. Some of the diseases for which stem cell research is projected to benefit are: heart disease, spinal cord lesions, non-union of fractured bones, Parkinson disease, Huntington disease, type 1 diabetes, corneal & retinal lesions, motor neuron disease, cerebrovascular disease, Alzheimer disease and muscular dystrophy (2).

One of the advantages of the use of laser light for investigating biological materials, particularly stem cells, is that it promotes limited use of reagents and chemicals that can interfere with the physiological properties of these therapeutic cells. More work on optical manipulation of stem cells further endorses the fact that there is normally minimal requirement for use of chemicals during optical experiments used in studying and answering biological questions. Uchugonova et al, 2008 (3) investigated the two-photon excited autofluorescence of multipotent human stem cells and the onset of collagen production of differentiated cells through detection of second harmonic generation signals at 435 nm. In this paper they also report that multiphoton microscopes hold novel non-invasive ability for marker-free optical stem cell characterization. Further in their other paper they reported on the optical cleaning of stem cells based on highly precise multiphoton processing using ultrashort near infra-red fs laser pulses. This was performed to isolate single cells of interest in order to inactivate undesirable single cells within three dimensional stem cell clusters (4). Thus, the ability to photo-transfect embryonic stem cells is highly desirable, especially because this transfection technique promotes the introduction of chemical-free naked pDNA.

The two major kinds of mammalian stem cells comprise of embryonic stem cells (from embryos) as well as adult stem cells (mainly from bone marrow). Generally embryonic stem cells are able to differentiate into all of the different specialized embryonic tissue. On the contrary, adult stem cells act towards the body’s repair system, replenishing specialized cells as well as maintaining the normal turnover of regenerative organs such as blood, skin, intestinal tissue, etc. Stem cells vary in the diversity of their differentiation descendants and are classified as totipotent (zygote stem cell), pluripotent (embryonic stem cells), multipotent (adult stem cells) (5). During my studies I made use of

express a foreign plasmid and were also optically induced to differentiate into a new cell type.

In both in vivo and in vitro investigations preservation of mainly pluripotent stem cells is of critical importance. As a result, recent literature reports on the dedifferentiation techniques allowing the reprogramming of fully differentiated cells into induced pluripotent stem cells that closely resemble ES cells in their developmental potency. Nuclear replacement technology (figure 6.1 (B)) is one way of achieving this but Takahashi et al, 2006 (6) demonstrated the induction of pluripotent stem cells from mouse embryonic and adult fibroblasts by introducing (via retroviral transduction) into these cells four pluripotency transcription factors known as Oct3/4, Sox2, c-Myc and Klf4 under ES cell culture conditions. These four and various others (e.g. Nanog) are crucial factors that are necessary to maintain pluripotency and a key feature reflecting the developmental capacity of ES cell lines is that in early embryo they express such markers of pluripotency. Therefore core transcription factors are required and essential to maintain the undifferentiated state in ES cells. These factors activate genes that are necessary for ES cell survival and proliferation while repressing target genes that are activated only during differentiation.

Stem cell based-therapy, their properties, differentiation pattern and lineage commitment are all essential biology topics currently under significant investigation in literature. However, there still remains a pressing necessity to answer the biological questions concerning how the renewal and differentiation programs are operated and regulated at the genetic level. Genetic manipulation such as delivery of exogenous gene expression or knockout with small interfering RNA (siRNA) is relatively rare in ES cells. The ability of ES cells and adult stem cells to differentiate into specific cell types holds immense potential for therapeutic use in cell and gene therapy. Realization of this potential depends on efficient and optimized protocols for genetic manipulation of stem cells.

The remainder of this chapter presents experiments where I investigated photo- transfection of pluripotent stem cells and also optically induced differentiation of these cells.