Cells develop from only one fertilized egg --- zygote. A "Totipotent Cell" with the ability to differentiate into more than 220 cell types. except placenta. But its Totipotency, (which represents the cell with the greatest differentiation potential) is not retained during all the various stages of cell sub-division.

The first sub-division provides 2 Totipotent cells. The second 4 cells, retain Totipotency But the following 8 cells no longer retain the ability to differentiate into all types of cells. Further sub-divisions differentiate into specialized cells such as fibroblasts, erythrocytes, nerve cells, intestinal mucosal epithelial cells and pancreatic islet cells, etc. but following 6 to 7 full further cell sub-divisions (about 5 days after fertilization), the embryo becomes a "Blastocyst" and possesses "Inner Cell Mass (ICM) "

ICM cells can differentiate into any cell type, except placenta --- pluripotency. 3 weeks after fertilization, the cells of the inner cell mass differentiate into ectoderm, mesoderm and endoderm. In the 1950s, this differentiation was thought to be irreversible, being described as a ball rolling down a hill and technically known as " Epigenetic Landscape".

This was until 1962 when John Gurdon created clone frogs and then in 1996 Ian Wilmut created "Dolly " the cloned sheep. Scientists had previously thought stem cells could not have pluripotency once dispersed into a specialized cell. however, in nature, even once differentiated, some cells can again differentiate and this phenomenon is known to science.

The stem cells of humans have limited ability to differentiate. But for example, Planaria that is a small flatworm living in rivers has amazing ability to differentiate into any organs, even into itself. When it is chopped into 3 fragments like picture, each fragment regenerates to a perfect Planaria. There is a record that 1/279 fragment of one Planaria regenerated to perfect Planarias. When Planaria grows to a certain size, it divides itself to two fragments. And each fragment grows to a perfect Planaria. For Planaria, division and regeneration are mechanism for reproduction. But under some circumstances Planaria chooses sexual reproduction.

A Plant can regenerate itself from one already differentiated cell. An Enzyme mixture of Cellulase Onozuka R-10 and Macerozyme R-10 dissolves tobacco plant (Nicotiana tabacum) leaf cell walls, and cells become leaf protoplasts (= cell without cell walls). When protoplasts are cultivated under appropriate condition, the protoplasts grow to a seedling plant through plant callus (= mass of unorganized parenchyma cells). When the seedling plant is planted in soil, it grows to a perfect tobacco plant.

Two British scientists proved biological technique could change Epigenetic Landscape.

Gurdon created cloned frogs.

Wilmut used a cell nucleus which had been differentiated in the mammary gland.

Human embryos reach "Blastocyst" about 5 days' post fertilization. The Blastocyst possesses an "Inner Cell Mass (ICM) " In 1981 Martin Evans succeeded in culturing mouse ICM cells. These cells are capable of propagating themselves indefinitely in an undifferentiated state, and they can differentiate into any type of cell except placenta. These cells are pluripotent, and known as Embryonic Stem Cell (ES Cells). James Thomson created ES Cells from Monkey embryo in 1995 and later in 1998 created ES Cells from human embryo. However, research into ES Cell needs further study due to an ethical dilemma, that in order to isolate inner cells from the blastocyst, the blastocyst is destroyed, so is the embryo at pre-implantation stage to be considered human? and even if not, do we have the right to destroy human potential growth.

The ethical issue of ES Cells can be by-passed by using Induced pluripotent stem cells. The iPS cell is a pluripotent stem cell which can be generated directly from adult cells, not from human embryos, and in 2006, Shinya Yamanaka and his team in Kyoto University created iPS cells from mouse fibroblasts.

He hypothesized that the genes playing a pivotal role in the function of ES Cells could induce an embryonic state in adult cells. But how many are the genes? The human has 20 - 25 thousand genes. Yamanaka researched and found 24 genes which were important for the characteristic protein of human ES Cells, and then used retroviruses to deliver all 24 genes into mouse fibroblasts, and the fibroblasts were able to propagate indefinitely. These iPS Cells were pluripotent like ES Cells.

Yamanaka removed one factor at a time from the 24 factors to identify the necessary genes for reprogramming and by this process he identified 4 factors -- Oct3/4, Sox2m Klf4 and c-Myc - named the "Yamanaka Factors", and Later he found c-Myc was not needed for reprogramming, but without c-Myc the process took longer and was inefficient.

A strong concern of the iPS researchers was if iPS Cell differentiation caused cancerous cells. But this issue has almost been resolved completely through rigorous study. And many clinical human applications are now carried out in Japan. For example, in 2014 retina transplantation by iPS Cells was successfully carried out for age-related macular degeneration. And cells did not differentiate into cancer cells. In my next page, I write more about these applications.

iPS Cells are useful not just for regenerative medicine but for drug discoveries or development. Because it is very easy for researchers to recreate special cells which cause special diseases, in a petri dish --- Alzheimer's disease, Parkinsons disease, ALS (Amyotrophic Lateral Sclerosis), Schizophrenia.

For example, "Achondroplasia" which is caused by mutation in fibroblast growth factor receptor 3. This is a common cause of dwarfism. Researchers made iPS Cells from skin fibroblasts of 3 patients with achondroplasia then allowed the iPS Cells to differentiate into chondrocytes over 2 to 3 weeks. Chondrocytes in the petri dish secreted about themselves an extracellular matrix which is characteristic of chondrocytes and made a mass. Compared to the chondrocytes of healthy people, these patients chondrocytes grew slowly, and the researchers tried thousands of drugs one by one to cure the abnormal chondrocytes from the petri dish specimens. Then finally, and with much surprise, they found the "Statin drug" was effective and able to cure the abnormal chondrocytes of achondroplasia patients. Why surprise? Because Statin drugs are for lowering cholesterol, and nobody expected cholesterol lowering drugs to be effective against achondroplasia.

In the next pages I explain in more detail about practical and clinical uses of iPS Cells.

The researchers of iPS Cells were most afraid of differentiation of iPS Cells into cancer cells. But now this problem has been almost solved by rigorous studies. And many clinical applications for humans are being done in Japan. For example, in 2014 transplantation of iPS Cells of retina was successfully done for age-related macular degeneration in Japan. The cells have not differentiated into cancer cells. In the next page, I write more about the applications.

iPS Cell is very useful not only for regeneration medicine but also for drug discovery or drug development. Because it is very easy for researchers to make the special cells that cause the special diseases --- Alzheimer's disease, Parkinson's disease, ALS (Amyotrophic Lateral Sclerosis), Schizophrenia -- in petri dish.

For example, Achondroplasia that is caused by a mutation in fibroblast growth factor receptor 3. This is a common cause of dwarfism. The researchers made iPS Cells from the skin fibroblasts of the 3 patients of achondroplasia and let the iPS Cells differentiate into chondrocytes in 2 to 3 weeks. The chondrocytes in petri dish secreted around themselves an extracellular matrix that is characteristic of chondrocytes and made a mass. The chondrocytes from the patients grow very slow compared with the chondrocytes from those of healthy people. The researchers tried thousands of drugs one by one to cure abnormal chondrocytes from the patients in petri dish. Then finally, and with much surprise, they found "Statin drug" was effective to cure the abnormal chondrocytes of achondroplasia patients. Why surprise? Because Statin drugs are drugs for cholesterol lowering. Nobody expected cholesterol lowering drugs are effective for achondroplasia.

In the next pages, I explain in more detail about practical and clinical use of iPS Cells.

Adult Stem Cells are undifferentiated cells found throughout the body such as in bone marrow, and umbilical cord blood, and the mammary gland, and the surface of the small and large intestines, the adipose tissue, the lining of the nose, the testicles, and the hair follicle, between the basement membrane and the sarcolemma of muscle fibers (Satellite Cells), etc.

These cells are multipotent cells that have less ability to differentiate into specialized cells than pluripotent cells. The adult stem cell from the bone marrow, called Hematopoietic Stem Cell (HSC), was discovered in the 1960s by two Canadian biologists, James Till and Ernest McCulloch, and has been used clinically to cure various blood diseases, such as leukemia, malignant lymphoma, multiple myeloma, etc. Clinically a very important cell. But for regenerative medicine, it needs much practical work to obtain stem cells from bone marrow, and requires general anesthesia, however scientists recently have found it easier to obtain these cells.

This is by ASC (adipose-derived stem cell) from our fat. The first scientific reports on ASC were made by an American scientist, Patricia Zuk (UCLA), in 2001. She reported the presence of mesenchymal stem cells in the fat tissues, and as they have a faster growth rate, these cells are expected to be advantageous for regenerative medicine.

ASC can differentiate into muscle, bone, cartilage, liver, adipose cell (lipid cell). And besides the advantages as stem cell, ASC secrets exsosome (nano size particles) that contain enzymes which dissolve beta-amyloid of Alzheimer's disease. The efficacy is 8 times more potent than the enzymes secreted by the exssome of the bone marrow.

ASC is now aggressively researched in Japan for practical uses. It will be used to treat many diseases such as Alzheimer's, Parkinson's, and diseases of the liver and kidneys, and periodontal disease, and more. For example, please see the video:

In Japan Tottori University Medical School researchers have established the technique of breast reconstruction by ASC after mastectomy due to cancer. They operate and inject ASC into the patients depressed breast. The breast recovers to the original shape within three months. This is not silicon, but the patients own cells. Quite natural. No rejection. The cost of this treatment will be covered by health insurance within three years in Japan. And Doctors at the Nagoya University Medical School use ASC against urinary incontinence stress. The sphincter function of the urethra often weakens due to aging,delivery, and some bladder diseases. ASC is injected around the patients urethra to strengthen the smooth muscle.

However, some side effects may occur by use of ASC. For example, male prostate hyperplasia and female endometriosis. Issues not studied in depth. So for now until the side effects have been dealt with, we should wait for general anti-aging treatments.

Recently it was found possible to induce, directly from somatic cells, not only iPS but nerve cells, hepatocyte cells, myocardium cells, cartilage cells, and many varied cells by introducing the specific key transcription factors in cell differentiation, which means that through by-passing of pluripotent stem cells it is possible to induce differentiated specific cells from somatic cells. This is called "Direct Reprogramming", and the most exciting research for example is: myocardial reprogramming in vivo, where Doctors inject patient's fibroblasts with transcription factors to the infarcted lesion of the heart. There the fibroblasts differentiate into myocardium (heart muscle). So, simple and quick. And this regenerative medical technique is under development mainly in Japan and the United States and as this technique is established, lots of heart surgeries will become obsolete and this is also true of brain surgery. The numbers of hospitals will eventually reduce and cost of time consuming surgeries will lower and this will lead on to a "De-Hospitalized Society".

For a basic understanding of stem cell mechanisms: ES Cell is studied along with iPS Cell and ASC. But ES Cell has an ethical dilemma and this issue shall not be overcome by science. So, it cannot be used for the treatment of human diseases. Direct Reprogramming: Wonderful technology. But it may take 10 or 20 years more to accomplish it. Therefore, at this moment, iPS Cell and ASC are most realistic medical tools for those who are suffering from degenerative diseases and wish rejuvenation.

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Stem Cell | Makise Medical Center Japan