Stem Cells for Cell Replacement Therapy
Said AL Hakmani
Parkinson’s disease is a movement disorder that results from damage to the neurons in the brain that produce the chemical named dopamine. The dopamine sends signals to parts of the brain that controls movement, allowing movement of smooth muscles. In Parkinson’s disease there is loss of the dopamine neurons. Symptoms of the disease include abnormal movement and tremor when a significant amount of dopamine has been lost. It is still not known exactly what causes Parkinson’s disease and the detailed pathophysiology is an area of active research (1, 13).
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Parkinson’s disease is of progressive nature, which implies that it deteriorates with time. But this happens gradually over numerous years. On the other hand, researchers are doing a great deal of exploration to better comprehend what is occurring in the cell. Some studies show that Parkinson disease is associated with an abnormal gene in some individuals, however so far there is insufficient evidence to demonstrate that it’s genetically inherited (1). Braak’s hypothesis recommends that Parkinson’s disease starts in the digestive tract and in the brain’s centre of smell. The hypothesis is upheld by the way that side effects connected with digestion and smell happen at an early stage of the disease. Professor Jia- Yi Li’s research team has now possessed the capacity to tack this methodology further, from the gut to the brain in rat models. The experiment demonstrates how the poisonous protein alpha-synuclein is exchanged starting with one cell, then onto the next before arriving at the brain’s movement center, leading to the characteristic movement impairment seen in Parkinson’s disease. He proves that disease process really can go from the peripheral nervous system to the central nervous system, in this situation from the wall of the gut to the brain (3, 23).
Usually people start to have symptoms of Parkinson’s disease between the ages of 50 and 60 years. But in certain cases it starts earlier.
The first presenting symptom of Parkinson’s disease is tremor (shaking or trembling). It affect the hands, arms, or legs. It is one of the most common signs, but not everyone has it. Also, not everybody with a tremor has Parkinson’s disease. The tremor often starts only on one side of the body or just one arm or leg. In addition, Parkinson’s disease can cause stiff muscles all through the body, so it causes problems like inconvenience gulping or constipation. In the later phases of Parkinson’s disease a person may have fixed expression, trouble talking and other problems. Some individuals lose mental skills (1, 22).
Figure 1 shows the nerve cells in the brain (1)
The power of stem cell
Stem cells have the potential to develop into various cell sorts in the body during life and schedule growth. In many different tissues they serve as a sort of internal repair system, dividing without limit to replace other cells as long as the animal or person is alive. At the point, when a stem cell divides, every new cell has the capacity either to remain a stem cell or get to be an alternate kind of cell with a more specific function, for example, a red blood cell, a muscle cell, or a brain cell and other cells (5).
The stem cells are featured from other cell types by two essential characteristics. First, they are capable of renewing themselves through cell division, frequently after long periods of inactivity. Second, under some physiologic or trail conditions, stem cell can become tissue- or organ-particular cells with special functions. In particular organs, for example, the gut and bone marrow, they consistently separation to repair and supplant exhausted or harmed tissues. Moreover, in diverse organs, for example, the heart and the pancreas, stem cell just partition under exceptional conditions (5, 20).
Researchers fundamentally worked with two sorts of undeveloped cells from animals and human: embryonic stem cells and non-embryonic stem cells.
Researchers in 1981 found approaches to get embryonic stem cells from early mouse embryos. In 1998 the definite investigation of the science of mouse undifferentiated cells prompted the revelation of human stem cells. However, these cells are called human embryonic stem cells. The embryos utilized in these studies were the result of invitro fertilization strategies for reproductive purposes. When they were no more required for that reason, they were donated for research with the informed consent of the donor (5, 21). Scientists made an alternate achievement by distinguishing conditions that would permit some particular adult cells to be “reprogrammed” to accept stem cell-like state in 2006. This new sort of stem cell, called induced pluripotent stem cells (iPSCs) (5).
These IPS cells may be used in the future to make the patient’s own cells, a safe cell source that is free from other factor which affects the patient. Animal studies in rat and mouse have been carried out to test the possibility of replacing the lost neurons and results have shown that these cells can develop into dopamine neurons. In human studies, it is possible to measure the increase of dopamine neuron function in the striatum of the patient by using positron emission tomography (5).
The Stem cells are essential for living organisms for different reasons. In the three to five days old embryos, called a blastocyst, the internal cells give rise to the entire body of the organism, including all of the many specific cell sorts and organs for example, the lungs, heart, skin and other tissues. In certain adult tissues, such as bone marrow, brain, and muscle, the adult stem cells create replacements for cells that are lost through ordinary injury, or disease. The stem cells make new potentials for treating diseases such as heart disease and Parkinson disease. However, much work remains to be carried out in the research facility to understand how to use these cell therapies to treat disease (5).Research on stem cells continues to advance about how can an organism develops from a single cell and how can healthy cells replace damaged cells in adult organisms. The stem cell research is one of the fascinating areas in contemporary biology (5).Many researchers believe that embryonic stem cell could be an excellent source of dopamine neurons because they can grow in a culture dish and make into any type of cell after long time in culture. Now, the dopamine neurons have been generated from the Human embryonic stem cell. One research study used the special type of companion cell with specific growth factors to promote the differentiation of the embryonic stem cell through several stages into dopamine producing cell (5).
Growth of the Human Embryonic Stem Cell
To grow the human embryonic stem cell, the cells will grow in the culture dish and will spread and divide on the surface of the media. The culture dish contains the mouse feeder cell which provides the nutrients into the culture media to grow the cell. However, researchers now devised a method to grow the embryonic stem cells without mouse feeder cells to avoid the risk of transfer of viruses from the media to the human (5,6 ,18).
The tests used to identify embryonic stem cells in the laboratory are lacking. But, the laboratories that grow the human embryonic stem cells use different types of test such as:
- Growing and subculture of the stem cell for many months to make sure of the health of the cell.
- The use of techniques to determine the presence of transcription factors that are produced by undifferentiated cells. There are two important transcription factors Nanog and Oct-4. Both factors help the genes to turn on and off to maintain stem cell undifferentiation and self-renewal (5, 24).
Conversion of human ES and iPS cells
One study of culture of stem cell found that the effectiveness of PSCs mouse derived DA neurons have shown efficacy in the models of Parkinson Disease. The developing midbrain is characterized by expression of the floor plate (FP) marker FOXA2 and the roof plate marker LMX1A. Canonical WNT signalling is critical for both roof plate function and midbrain DA neuron development. WNT activation incite LMX1A expression and a neurogenic change of PSCs derived midbrain FP towards DA neuron fate. Exposure to CHIR potent GSK3B inhibitor known to strong activate WNT signalling induce LMX1A in FOXA2 and FP precursors. CHIR is more potent than recombinant WNT3A or WNT at inciting LMX1A expression. The efficiency of LMX1A induction depends on the time of CHIR exposure it effect from 3-11 days. Thus, CHIR is the most critical factor for inducing coexpression of FOXA2/LMX1A. FOXA2/LMX1A coexpression require strong expression of SHH signalling using purmorphamine small molecule agonist. Treatment with SHH agonist and FGF8 in the absence of CHIR show significant lower expression of FOXA2 by day11 and complete absence of LMX1A expression. An expression of, NGN2, LMX1A and DDC showed establishment of midbrain neuron precursor fate already by day11. Both midbrain FP and DA neuron induction ready to reproduce in independent ES cell and human induced PSCs line. In culture TH positive cells showed coexpression with NURR1 only in FP derived DA neurons coexpressed FXOA2 and LMX1A. Few GABA and serotonin can observe. But, DA level present in culture about eight times higher in FP (9, 12, 15, 19).
There are several lines of evidence to demonstrate a crucial role for SMAD signalling during neural induction. Studies in frog identified bone morphogenic protein (BMP) inhibitors, including, follistatin, chordin and noggin as the basic neural affecting factors in the Spemann organizer (10).
The drug SB431542 was demonstrated to improve neural prompting in an embryoid body-based hES cell neural induction protocol. SB431542 inhibits the Lefty/Activin/TGFβ pathways by blocking phosphorylation of the ALK5, ALK4, and ALK7 receptors. In addition, Noggin or SB431542 treatment improves the neural induction and blockade the SMAD signalling to achieve full neural conversion. The hES cells were exposed for 72 hours to the drug SB431542 or Noggin and observed the growth factor of neural induction was monitored by expression of PAX6 earliest marker of neuroectodermal differentiation. The combined treatment with both Noggin and SB431542 increased the efficiency of neural induction to more than eighty percent of total cells , compared with less than ten percent PAX6 cells when used Noggin or SB431542 alone(10, 17).However, the only PAX6a converts the human embryonic stem cell to neuroectoderm (11, 16).
Figure 2: show the model of proposed mechanisms contribute the action of Noggin and SB431542 (10).
From the diagram above at the higher density, essentially CNS cells that are PAX6 are formed which are equipped of giving rise to R-NS cells and example capable neuronal populations of motoneurons and dopaminergic neurons inside 19 days of differentiation. While at lower densities both CNS fates and neural crest fates are observed. The neural crest lineages include melanocytes and the neural crest precursor cells amenable to designing and subtype determination reactions (10, 18).
In this figure3 shows one research on mice how the people try to make new dopamine. Isolated ventralmidbrain cells were expanded and patterned in vitro before to transfection to overexpress WNTs. The cell phenotype was examined following in the vitro differentiation or transplantation into Parkinsonian mice. Morphogens SHH and FGF8 significant increase the proportion of TH positive spheres out of total spheres compared with FGF2 treatment alone (13).
Dopamine is a chemical signal that transfers information from one neuron and the next neuron. The receptors found on the cell surface bumps the signals down to the receiving neurons. The dopamine interested in many different important pathways and it found primarily in the mesolimbic pathways which starts with cells in the ventral tegmental area in the midbrain (2).Dopamine plays a number of roles in human and other animals. It controls many functions such as movement, memory, pleasurable reward, attention, sleep, learning, mood, inhibition of prolactin production, and behaviour. The increase and deficiency of this dopamine chemical are the cause of several diseases like Parkinson’ disease. In the brain, part called the basal ganglia controls movement. Basal ganglia thus rely on upon a certain amount of dopamine to function. The action of this occurs by dopamine receptors D 1-5. The deficiency in dopamine in the brain may become delayed and uncoordinated the movement and other function of the body (3).
The dopamine is an important neurotransmitter in the brain. Substantial part of the overall dopamine in the body is produced outside the brain by mesenteric organs. The production of dopamine within the central nervous system, depend on the dopamine biosynthesis. There are two steps of dopamine biosynthesis takes place in the cytosol of CAergic neurons and starts with hydroxylase of L-tyrosine at the phenol ring by tyrosine hydroxylase to produce DOPA(4, 14).
Figure4 shows the neuronal dopamine metabolism(4).
This figure5: show how process of the fibroblasts are taken to culture to induce neural stem cells to make dopamine neurons and transfer to the patient to functional recovery (25).
Good Manufacturing Practice
There are many challenges to the use of stem cells for replacement therapy. However, the good manufacturing practice (GMP) is a quality assurance system that is used to make sure the product of stem cells is good and safe to use for treatment. The GMP in human embryonic stem cell culture required several components for good clinical practice. First, the culture or the feeder media that use in the laboratory must be reached with standard of GMP. That is why so many are now trying to grow these cells in feeder free conditions. Finally, all components of the culture and cryopreservation media should be validated for use in the GMP system and be non animal derived products (7).
The ethical issue surrounding the use of embryonic cell need be carefully controlled. In UK human embryonic stem cell can gathered from left over embryos produced as part of in vitro fertilisation. This can done under strict UK guidelines from Human Fertilisation Embryology Authority (HFEA) and the human tissue authority (HTA) only in the early stage embryos can be used in research up to a maximum of fourteen days of development(8).
In my conclusion, the development of the research in the recent year will found the treatment of some diseases that affect the human in the neuron system like Parkinson’s disease. In addition, the study of stem cell research may treat disease by transplanted the cell to the body of the human, but the risk factor that makes this study difficult is the potential of the stem cell growth. The study of stem cell factor may solve the problem of cell growth. Also, the feeder that use to make culture is important to grow the cell without any contamination.
There are many factors that help the stem cell to grow in the culture dish. These factors make stem cell to grow fast and convert to the different type of tissue organs. Also, there are some inhibitors that inhibited the factors when stem cells are converted to the different sorts.
The research on the stem cell still under the process of trail. It is successful to treat some disease, but in other disease is still trying in animal model if it’s successful may try with human, those have Parkinson’s disease.
|2||iPSC||Induced Pluripotent Stem Cell|
|3||PSC||Pluripotent Stem Cell|
|4||HESC||Human Embryonic Stem Cell|
|6||FOXA2||Forkhead box protein A2|
|7||LMX1A||LIM Homebox transcription factor 1 Alpha|
|8||WNT||Group of signal transduction pathways made of proteins|
|9||OCT-4||Octamer binding transcription factor 4|
|11||CHIR||Chemical High selective for GSK3 inhibitor|
|12||GSK3B||Glycogen synthase kinase 3 Beta|
|13||WNT3A||One Activator of WNT pathway|
|15||FGF8||Fibroblast Growth Factor 8|
|16||iES||Induce Embryonic Stem|
|20||NURR1||Nuclear Receptor Related 1 protein|
|21||GABA||Gamma Aminobutyric Acid|
|22||SMAD||proteins are versatile intracellular mediators of those signals|
|23||BMP||Bone Morphogrnic Protein|
|24||SB431542||Drug inhibitor of the activin receptor-like kinase receptors, ALK4, ALK5 and ALK7.|
|25||TGFβ||Transforming Growth Factor Beta|
|26||ALK4,5,7||Activin receptor Like Kinase 4,5,7|
|27||PAX6||Paired box 6 Gene|
|28||CNS||Central Nervous System|
|29||R-NS||Rosette Neural Stem|
|31||GMP||Good manufacturing Practice|