Can Skin Dermal Papilla (DP) Cells Differentiate into Induced Pluripotent Stem Cells?

Pluripotent stem cells are the stem cells that can only differentiate into a limited range of differentiated cells. (2) They have the ability to give rise to all somatic cells from ectoderm, mesoderm and endoderm, as well as gametes. Naturally it can be found in embryos as Embryonic stem cells (ES cells). Induced pluripotent stem cells (iPS cells or iPSC) are the pluripotent stem cells that are generated directly from adult cells, first discovered by Shinya Yamanaka in 2006 by using a set of reprogramming factors (Oct4, Sox2, Klf4, and c-Myc or LIN28 and Nanog) (3) to reprogram mature cells back to a pluripotent state (4).

mEFs can either be purchased commercially or obtained in the following manner. Pregnant mice are sacrificed between 12,5 and 13,5 days after mating. Embryos are removed from uterus and placed in sterile PBS. Head and internal organs are removed from embryo. In fresh PBS 1mm sections are cut then transferred into trypsin/EDTA. Stir cells for 40 minutes, add DNase if it looks viscous and clumpy. Proceed by adding mEF medium, then strain the solution

Keywords: ethical, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), disease, drug development, research

Human embryonic stem cells (hESCs) are pluripotent and are obtained from the inner mass of a 4-5 day old human blastocyst that consists of approximately 100 cells (“Stem cell research,” 2009).

The creation of induced pluripotent stem cells by direct reprogramming has allowed for the circumvention of using embryonic stem cells while still leaving the cells with the ability to maintain pluripotency. Instead of ES cells which were originally derived from the epiblast of mouse embryos, IPS cells were generated from mouse embryonic fibroblasts. This eliminated both any ethical concerns for whether those cells were a living being or not and the need to destroy embryos at the blastocyst stage. An advantage of IPS cells is that they are derived from human somatic cells which makes them easy to acquire due to the possibility of using skin or blood cells. They can also be grown and differentiated individually for each person that the sample of somatic cells is taken from which eliminates the possibility of having any immune reaction and rejection to the differentiated cells during transplantation. These characteristics of IPS cells are important because they are what enables us to safely and accurately transform these affected cells from patients cells into neurons and confidently study them.

Those people also argue that society would begin to accept the concept or destroying one potential life in order to save another. The final source of embryonic stem cells is opposed only because people find creating an embryo only to destroy it later is morally wrong and inhumane. Those that are in favor or neutral to creating the embryos point out that it is acceptable for an embryo that is created accidentally to be destroyed as a consequence of pregnancy, but to create an embryo with the intent to use the stem cells it is composed of to save a life is wrong (Hug). The second type of stem cell are pluripotent stem cells. Pluripotent stem cells come from fetal tissues instead of the embryo itself. Fetal tissues include the umbilical cord and the blood inside it, and the placenta. There is also a second kind of pluripotent stem cell. These stem cells are called induced pluripotent stem cells, or iPSCs. iPSCs are stem cells were adult stem cells, but have been reprogrammed into their embryonic stages. Though iPSCs resemble human embryonic stem cells, they are not usually accepted into the body, and so they are introduced to other adult stem cells with a virus that has caused some cancers to develop (National Institutes of Health).

The ability to manipulate the stem cell corresponding to a specific organ/tissue remains important. A type of stem cell that can be manipulated is the embryonic stem cell. These stem cells descend from embryos aging from three to five days (Watt) (Driskell). During earlier stages, scientists describe embryonic stem cells as “blastocysts” which contain over one-hundred and fifty cells (Watt) (Driskell). They duplicate into more cells or transform to any cell located in the body (Watt) (Driskell). This “duplication” allows embryonic stem cells to regenerate and repair diseased tissues. Embryonic stem cells gain importance in cancer treatments—if doctors diagnose patients with leukemia, then during chemotherapy, the doctor can infuse embryonic stem cells into the body. Since the cells are young, they can repair the targeted cell, aiding cancer treatments and the patient. In addition, this technique is used with another type of embryonic stem cell called “pluripotent stem cells”. Pluripotent stem cells originate as inner mast cells (cells

The human embryo for embryonic stem cell research requires the ova from a woman to make this possible. This requires many risks to the woman giving the egg. “Embryonic stem cells are pluripotent cells positioned in the early embryo” (Miller Ph.D., Levine Ph.D.). Pluripotent means that the cells are capable of developing into most of the body’s cell types and have the ability to aid and cure diseases. (Miller Ph.D., Levine Ph.D.). This pluripotency is what distinguishes between embryonic and adult stem cells. The embryonic stem cells can be generated in every cell type in the body and can indefinitely create themselves making it possible for tissue replacement in addition to finding cures for diseases. “Embryonic stem cells are human embryos that develop after fertilization into a blastocyst” (Miller Ph.D., Levine Ph.D.). Hundreds of immune system diseases and rare genetic disorders are believed to be among the possible to be aided or cured using embryonic stem cells. Embryonic stem cells

In the research that will be conducted at Harvard, if permission is granted, the growth of the blastocyst would be stopped and it would not be implanted into a woman’s womb. Stem cells would be extracted for study, destroying the embryo.

In 1981, Martin Evans of the University of Cambridge revolutionized the field of medicine when he became the first person to identify embryonic stem cells. In a study that involved observing cultures of mouse embryos, Evans and his team discovered cells that behaved much differently than adult somatic cells. These cells, derived from the inner cell mass of mammalian blastocysts, had the incredible property of pluripotency. They were undifferentiated and able to grow indefinitely into cells of all three germ layers (Evans 1981), a completely unique ability that had been previously unheard

During the earliest stages of the development of the embryo, its cells are entirely undifferentiated. When a cell has not yet discovered its operation in an organism, it is classified as a stem cell. The two major varieties of stem cells have been distinguished as pluripotent and totipotent. An example of a totipotent cell is an egg that has been fertilized by a sperm cell, and is a single cell egg. This cell now has the ability to develop into any and every human cell. “The first few cell divisions in embryonic development produce more totipotent cells. After four days of embryonic cell division, the cells begin to specialize into pluripotent stem cells” (Biomed Brown Edu). Pluripotent cells however, are not as adaptable. Nicknamed the “master cell”, pluripotent cells have the ability to produce cells from the three basic body layers, enabling the possibility for them to create all cells or tissues that the body may need to heal (Children’s Hospital Organisation).

This guide was therefore used, along with the microinjection, to target POU5F1 in human preimplantation embryos. CRISPR-Cas9 editing was performed on IVF zygotes that had been donated to test whether OCT4 is required in the development of human embryos. 37 zygotes were microinjected with sgRNA2b-Cas9 complex and 17 were injected with just Cas9 as a control for the microinjection. It was suggested that the microinjection did not affect the viability of the embryos as 47% of the control embryos developed into blastocysts which is equivalent to those of un-injected controls. Only 19% of embryos microinjected with sgRNA2b-cas9 developed to the blastocyst stage. The quality of the blastocysts that formed significantly varied. They all retained a thick zona pellucida and had a distinguishable blastocoel cavity however very few possessed a small compact inner cell mass. Most of the embryos injected with the complex didn’t develop into a fully formed blastocyst as they collapsed in on themselves and degenerated. These results suggest that the integrity of the human blastocyst is compromised because of OCT4

Human embryonic stem cells (ESCs) are pluripotent cells isolated from blastocysts, and are highly useful in studying human development (Itzkovitz-Eldor et al., 2000 p. 88). Although the National Institute of Health states that “it is not known if iPSCs and embryonic stem cells differ in clinically significant ways”, iPSCs are already being used to achieve the same results as ESCs in some applications without the use of embryos, removing the ethical concern associated with ESCs (National Institutes of Health, 2009). ESCs are capable of differentiating into all cell types, and can be used as a source of differentiated cells. In the report by Itskovitz-Eldor et al., they discuss the induced differentiation of ESCs in suspension into embryoid bodies, including the three embryonic germ layers. The authors state that “the ability to induce formation of human embryoid bodies that contain cells of neuronal, hematopoietic and cardiac origins will be useful in studying early human embryonic development” (Itzkovitz-Eldor et al., 2000 p. 88).

Induced pluripotent stem cells [iPS cells] are specialized adult somatic cells that have been genetically modified to function similarly to embryonic stem cells; that is, iPS cells have the ability to differentiate into all adult cell types (2). The discovery of iPS cells in 2006 was very exciting for the medical community as researchers hoped to use iPS cells in many ways, including: to gain an increased understanding of disease pathology, as aids in transplant medicine, and in pharmaceutical and cytotoxicity studies. Subsequent research on iPS cells has revealed some areas of concern including:

To achieve these objectives, we will use three different mouse embryonic stem (ES) cell lines (R1, D3, E14), already present in the lab. In ongoing studies, it was noted that these cells presented all five of the ING genes with altered expression when they were induced to differentiate. We will first study if altering ING5 and ING4 expression using siING5 and siING4 to decrease it and pCI-ING5, pCI-ING4 to increase levels, will affect differentiation and the self-renewal rate of these lines. We will use sphere formation assays to assess self-renewal. We will also induce differentiation with these different expression constructs, thus measuring the influence of these proteins in the process. To determine the degree of differentiation with varying expression of the ING genes, we will use flow cytometry to follow changes in stem cells markers, such as OCT4, OLIG2 and Nestin16.