Mulitcellular Organisms

Stem Cells (2022 Update)

Stem cells are primitive cells that give rise to other types of cells. Also called progenitor cells, there are several kinds of stem cells. Totipotent cells are considered the "master" cells of the body because they contain all the genetic information needed to create all the cells of the body plus the placenta, which nourishes the human embryo. Human cells have this capacity only during the first few divisions of a fertilized egg. In the Bush era research on human embryonic stem cell lines may be conducted with Federal support if the cell lines meet the President’s criteria under the executive order announced on August 9, 2001.

		There are 69 cell lines registered with NIH - National Institutes of Health, 15 of these are available for shipment. President Obama signed an executive order on March 9, 2009 to lift the ban under the Stem Cell Policy in the Bush era. He also directed the NIH to develop revised guidelines to ensure that such research "never opens the door to the use of cloning for human reproduction". Legally it is possible that Obama's successor can in turn reverse the Obama executive order. Now back to the stem cell:
Figure 10-13a Differentiation Pathway	Figure 10-13b Stem Cell Cycle [view large image]

After 3 - 4 divisions of totipotent cells, there follows a series of stages in which the cells become increasingly specialized. The next stage of division results in pluripotent cells, which are highly versatile and can give rise to any cell type except the cells of the placenta. At the next stage, cells become multipotent, meaning they can give rise to several other cell types, but those types are limited in number. An example of multipotent cells is hematopoietic cells - blood stem cells that can develop into several types of blood cells, but cannot develop into brain cells. At the end of the long chain of cell divisions that make up the embryo are "terminally differentiated" cells - cells that are permanently committed to a specific function. Figure 10-13a shows the pathway from embryonic stem cell to multipotent stem cell and on to the different type of specialized cells.

Multipotent stem cell is self-renewing. When the stem cell divides, one of the two daughter cells may go on to give rise

to other types of cell, whereas the other daughter cell remains a stem cell, capable of dividing again and always giving one daughter to diversification. (See Figure 10-14b)

Blood cells in human body are replaced every 120 days. The replacement comes from the multipotent stem cell in the bone marrow. Skin cells are shed every few weeks. The replacement comes from the multipotent stem cell at the base of the skin (the basal layer). Scientists are still looking for the the neural stem cell. Its identity, location and potential remain unclear.

It has been long held that differentiated cells cannot be altered or caused to behave in any way other than the way in which they have been naturally committed. New research, however, has called that assumption into question. In recent stem cell experiments, scientists have been able to persuade blood cells to behave like neurons, or brain cells.

Recent research in 2004 has located the neural stem cells in the subventricular zone (SVZ). It is a principal source of adult neural stem cells in the rodent brain, generating thousands of olfactory bulb neurons every day. These neurons migrate from the SVZ to the brain region concerned with sensing smell. But such stem cells have become non-functional in adult human. The systematic decrease in the extent of adult neurogenesis during vertebrate evolution may be the result of an adaptation to keep neuronal populations with their accumulated experience for an entire lifespan. Therefore, the therapeutic transplantation of new neurons to regions of the human brain that are responsible for more advanced brain functions may be counterproductive, but their transplantation to other regions, such as the sensory or motor systems of the brain, could have enormous clinical significance. The identification of the cellular and molecular mechanisms that prevent adult neural stem cells from becoming integrated into functional neuronal networks would be a major accomplishment for repairing brain damage or lost neurons.

The Nature magazine has kindly made available a poster (2.8 MB in pdf format), which provides an overview of distinct embryonic and adult stem-cell types.

A 2012 paper in Nature reported that Optic Cup (a rounded goblet of retinal tissue in the back of the eye) has been grown from

	stem cell culture (from mouse for now). The process involves carefully controlled external environment (such as number of stem cells ~ 9000 in this case, the concentration of growth factors, and the addition of cell-culture ingredient called Matrigel etc). After introducing a right mixture into the stem cells, the rest is up to themselves to run their internal programs without any other supporting tissue. The optic cup emerge naturally in about 24 days (see Figure 10-13c). This feat has obvious implication for repairing vision.
Figure 10-13c From Stem Cells to Optic Cup [view large image]	Further researches are expending into the areas of pituitary gland, cerebral cortex, a complete photoreceptors, and cerebellum, but not the whole brain - that would be enormously difficult and ethically fraught.

[2022 Update]

		Stem cells are similar to common cells. It contains a nucleus, mitochondria, endoplasmic reticulum, ribosomes, membrane ... (Figure 10-13d) - nothing out of the ordinary. What makes them standing out is their ability of differentiating into specialized cell type by cue (Figure 10-13e). As explained further in the followings, there are different types of stem cells according to their potency (ability) and different kinds of activation cues for activating different genes (to produce specialized cell type).
Figure 10-13d Stem Cell	Figure 10-13e Activation Cues [view large image]

Totipotent Cell - Totipotent stem cells are unique as they have the greatest developmental potential compared with other stem cells. Examples include :
- Spore - a unit of sexual or asexual reproduction for many plants, algae, fungi and protozoa (single cell organism).
- Zygote - a eukaryotic cell formed by a fertilization event between two gametes. The zygote's genome is a combination of the DNA in each gamete, and contains all of the genetic information of a new individual organism.
- Embryonic Stem Cells (ESC) - they contain all the genetic information needed to create all the cells of the body plus the placenta, which nourishes the human embryo. Human cells have this capacity only during the first few divisions of a fertilized egg (the zygote).
Pluripotent Stem Cells (PSC) - this is the embryonic stem cell which has all the properties of the totipotent cell except the ability of generating placenta. These cells have the ability to undergo self-renewal and to give rise to all cells of the tissues of the body as shown in Figure 10-13f. The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate induced pluripotent stem cells (iPSCs) with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research. See "Induced Pluripotent Stem Cells: Problems and Advantages when Applying them in Regenerative Medicine" for a thorough review of iPSC.

Multipotent Stem Cells - They have the ability to develop specific types of cells (terminally differentiated cells). Ordering according to the lineage (from endoderm, to mesoderm, and ectoderm) as shown in Figure 10-13f, examples include :

Intestinal stem cells (ISCs) - Self-renewal in the intestinal epithelia is fueled by a population of undifferentiated intestinal stem cells that give rise to daughter or progenitor cells, which can subsequently differentiate into the mature cell types required for normal gut function.

Mesenchymal stem cells (MSCs) - They can differentiate into a variety of cell types, including bone cells (osteoblasts), cartilage cells (chondrocytes), muscle cells (myocytes) and fat cells that give rise to marrow adipose tissue (adipocytes). They can be found in a variety of tissue including adipose tissue (fat), bone marrow, umbilical cord, blood, liver, dental pulp, and skin.

Hematopoietic stem cells (HSC) - They are immature cell that can develop into all types of blood cells, including white blood cells, red blood cells, and platelets. Hematopoietic stem cells are found in the peripheral blood and the bone marrow.

Epidermal stem cells - These stem cells reside in the epidermis and hair follicle to ensure the maintenance of adult skin homeostasis and hair regeneration, but they also participate in the repair of the epidermis after injuries.

Neural stem cells (NSCs) - NSCs primarily differentiate into neurons, astrocytes, and oligodendrocytes. They are self-renewing cells that generate the neurons and glia of the nervous system of all animals during embryonic development.Glial cells do not divide once it is differentiated. Nerve cells or neurons also don't divide generally. However, some neural

Figure 10-13f Stem Cell Types [view large image]

progenitor stem cells persist in highly restricted regions in the adult vertebrate brain and continue to produce neurons throughout life. A good example is the hippocampus which makes memory.

Those which are terminally differentiated and do not have any precursor left for their repair of any injury which may happen in the future, such as heart cells.
The second type of differentiated cells are those which are kept in the G0 phase and as and when they are required they are used for replenishing the dead cells such as skin fibroblasts, smooth muscle cells, the endothelial cells that line blood vessels, and the epithelial cells of most internal organs, such as the liver, pancreas, kidney, lung, prostate, and breast. Such process is called mitosis.
The third type of cells are those differentiated cells which include blood cells, epithelial cells of the skin, and the epithelial cells lining the digestive tract which have short life spans and must be replaced by continual cell proliferation in adult animals. They are replaced via the proliferation of multipotent stem cells.

List of human cell types derived from the germ layers

Followings are some of the stimulations (cues) interacting with various types of stem cells :

See "Spatial Cue" in very early embryonic development and "Chemical Cues" during the formation of an embryo.

Mechanical Forces - This cues involve physical factors such as force, geometry and matrix elasticity. They play critical roles in defining cell and tissue functions, morphology and regeneration as well as human physiology and pathology. At the cell level, cell traction forces generated from sensing and responding to the extracellular environments can consequently influence many biological processes such as wound healing, angiogenesis and metastasis (see detail in "Mechanobiology").

In the micro-environment, mechanical forces can influence processes such as differentiation of stem cells and metastasis of cancer cells. Mesenchymal stem cells (MSC) have been shown to commit to specific phenotypes depending on tissue-level elasticity. For example, soft substrates that mimic the brain are shown to be neurogenic, stiffer substrates that mimic muscle are myogenic, while rigid substrates that mimic bone are osteogenic.

Figure 10-13h Mechanical Cue [view large image]

Thus, daily activities such as physical exercise, massge, acupuncture, ... can also promote the gene expression in stem cell (Figure 10-13h).

Growth Factors - This kind can be illustrated by the growth of organoids (man-made tissue) in the lab. As shown in Figure 10-13i, pluripotent Stem Cells (PSCs) and adult stem cells (AdSCs = multipotent cells,) are guided toward the maturation of a specific organoid by the introduction in culture of specific growth factors that activate (arrow up, green) or repress (arrow down, red) particular signaling pathways. The growth factors include bone morphogenetic protein (BMP); epidermal growth factor (EGF); fibroblast growth factors (FGF); hepatocyte growth factor (HGF); insulin-like growth factor (IGF); microtubule associated protein kinase (MAPK); RHO-associated protein kinase (ROCK); transforming growth factor (TGF); vascular endothelial growth factor (VEGF); and wingless-related integration site (Wnt). See "The role of physical cues in the development of stem cell-derived organoids".

Figure 10-13i Growth Factor Cue [view large image]

Genetic Influence - Such mechanism has been studied mainly on Hematopoietic stem cells (HSC). It is found the its stem-ness is maintained by different molecular processes that are crucial for regulating quiescence and self-renewal. The stem cell niche provides the micro-environment to keep HSC in a quiescent state. Furthermore, several transcription factors and epigenetic modifiers are involved in this process. Figure 10-13j shows the stem cell niche provides signals that regulate HSC quiescence and localization in the niche, e.g., DL1

Notch; CXCL12

CXCR4; Wnt

Frizzled (receptor). HSC intrinsic transcription factors regulate signaling process to keep HSC in a quiescent stage (see details in "Genetic and Epigenetic Mechanisms That Maintain Hematopoietic Stem Cell Function").

Figure 10-13j Genetic Cue

Stem-cell Niche - It is an area of a tissue that provides a specific micro-environment, in which stem cells are present in an undifferentiated and self-renewable state (Figure 10-13k). Cells of the stem-cell niche interact with each other for maintenance or promoting their differentiation. It is a complex, heterotypic, and dynamic structure, which includes supporting extracellular matrix,

neighboring niche cells, secreted soluble signalling factors (such as growth factors and cytokines), physical parameters (such as shear stress, tissue stiffness, and topography), and environmental signals (metabolites, hypoxia, inflammation, etc.). Stem cells, blood vessels, nerves, matrix glycoproteins and the three-dimensional space forming this unit provide a highly specialized microenvironment. Contact and communication between these elements is critical for stem cell self-renewal and cell fate regulation, thus rendering tissue homeostasis and regeneration. Niche has been found in lungs, skin, vasculature (network of blood vessels), heart, liver, kidneys, intestinal crypts, gastric glands, endometrium, muscle, bone marrow, bone marrow, and hippocampus.

Figure 10-13k Stem Cell Niche

Electrical Cues - Electrical Stimulation (ES) can activate many intra-cellular signaling pathways, and influence intra-cellular micro-environment, as a result, affect cell migration, cell proliferation, and cell differentiation. It has been found that continuous and defined levels of electric current promote NSC proliferation by 2-fold; while pulsed electromagnetic field exposure could enhance MSC proliferation as well. Poly-electrolyte hydrogel matrices (charged polymer networks) can support long-term survival and enhance proliferation (1.5-fold) of human MSCs. Proper ES promotes cell proliferation, usually under continuous stimulation of < 1 V/cm. Within the ES intensity range the cell proliferation rate increases with increasing intensity. For examples, pre-osteoblasts (blast = germ layer), osteoblasts, unrestricted somatic human stem cells, human umbilical vein ECs, NSCs, human dermal fibroblasts exhibited 0.2 to 1.5 times proliferation (Figure 10-13l), with increasing cellular metabolic activity, and do not

	affect cell phenotype. High-intensity ES of > 100 V/cm is also favorable for cell proliferation in a short period (< 1 ms) single stimulation, but excessively high intensity leads to cell death (see for example : "Electrical stimulation as a novel tool for regulating cell behavior in tissue engineering").
Figure 10-13l Electrical Cue

Oxygen Tension - Hypoxia (low oxygen level ~ less than the 21% in ambient environment) maintains undifferentiated state of embryonic, mesenchymal, hematopoietic and neural stem cell phenotypes. It is found that MSCs cultured under hypoxia for approximately 2 weeks showed increased proliferation and viability. During long-term culture, hypoxia delayed phenotypic changes in MSCs (such as increased cell volume, altered morphology, and the expression of senescence-associated-ß-gal), without altering their characteristic immunophenotypic characteristics. Furthermore, hypoxia increased the expression of stemness and chemokine-related genes, including OCT4 and CXCR7, and did not decrease the expression of KLF4, C-MYC, CCL2, CXCL9, CXCL10, and CXCR4 compared with levels in cells cultured under normoxia. That said, hypoxia is harmful to the body as a whole (see Figure 10-13n).

Figure 10-13m Oxygen Tension

Figure 10-13n Normal O₂ Partial Pressure (PO₂) in Various Tissues .

In Figure 10-13m, HIF = Hypoxia Inducible Factor, ROS = Reactive Oxygen Species, HO-1 = Heme Oxygenase 1, Nrf2 = Nuclear Receptor Factor, GSH/GSSG = Glutanthione Ratio, OXPHOS = Oxidative Phosphorylation, LDH = Lactate Dehydrogenase, PDK = Pyruvate Dehydrogenase Kinase, MAPK = Mitogen Activated Protein Kinase, PI3K = Phosphoinositide 3 Kinase, ERK = Extracellular Signal Regulated Kinase, VEGF = Vascular endothelial Growth Factor, OSKM = Oct3/4, Sox2, Klf4 and c-Myc.
See "Relevance of Oxygen Concentration in Stem Cell Culture"

From a developmental point of view, the fact that adult niches remain at low oxygen pressures correlates with the PO₂ (partial pressure of oxygen) values recorded in embryos, where embryonic stem cells (ESCs) develop and give rise to cells of all three germ layers. The pre-implantation human embryo and blastocyst develop under relatively low oxygen concentrations in vivo, approximating 2–9% O₂. The effect of oxygen on pre-implantation embryos has been comprehensively examined in several species, including the human. While embryos are capable of developing under a 20% O₂ atmosphere, studies have demonstrated compromised embryo development and viability under these conditions.

Such low oxygen requirement could be related to the fact that life started in the absence of oxygen long before the advent of photosynthesis which makes oxygen and is actually poisonous to early life (see Figure 10-13o). Somehow, the incorporation of ATP producing mitochondria in the eukaryotic cell to generate energy has the problem resolved and life proceeds to develop with some lingering relics. This process (and its reverse) is summarized by one single chemical equation :

Figure 10-13o Early Atmosphere

Xenobot - By 2020, the African Frog, Xenopus has become valuable model organism to study early embryonic development as well as

		for research into human genetic disease (complementary to the more commonly used mouse and zebrafish). Recently, it is found that the stem cells extracted from the early embryo can replicate itself (touted as a new way of reproduction - the so-called "kinetic replication" ???). Closer examination reveals that it can be explained by the various cues (as presented above) on the stem cells.
Figure 10-13g2 Xenobot, Stem Cells Extraction	Figure 10-13g3 Xenobot, Replication

In the "xenobot experiment", stem cells are extracted at the stage before gastrulation from an egg with about 4000 stem cells. These cells are about to differentiate into the 3 derm layers, i.e., the ectoderm, mesoderm and endoderm, according to a switch called EGA (Embryonic Genome Activation). Thereafter, development would come under the exclusive control of the zygotic genome rather than the maternal (egg) genome. See pictorial illustration in Figure 10-13g1 below :
Figure 10-13g1 Early Embryonic Development of Xenopus [view large image].

This kind of disruption has a fatal consequence for the fate of the stem cells, i.e., they become free to develop in whichever way provoked by cues within the cell mass and from the environment - something like a "headless chicken" as shown by the video in Figure 10-13g2 (click ), which lists its activities including swimming, running around in maze, tunneling in narrow capillary, interacting with particles, and self-repairing when injured, ...

Among all such activities, it is found that such collection of stem cells can replicate. The Xenobot experiment is to enlist the help of Artificial Intelligence (AI) which tested billions of body shapes (see some of the shapes ) to make the xenobots (bot is now appended to indicate it is something like a robot) more effective at this type of replication. As shown by the video in Figure 10-13g3 (click ), the C-shaped "bot" is the most efficient earning the title of "The World's First Self-replcating Living Robot".

Instead of treating it as a new type of reproduction, the replication of Xenobot can alternatively be explained as the result of picking up various cues interacting with the stem cells, for example :
- Spatal cue to minimize energy should be responsible for the initial spherical shape.
- The running around in maze, tunneling in narrow capillary, ... should be under the heading of mechanical cue. It is provided by Newton's Laws : 1. velocity (v) = constant, acceleration (a) = 0, for force (F) = 0, 2. F = ma, and 3. reaction = action.
- The crucial factor could be the tendency of the stem cells to form "niche", which is a complex, heterotypic, and dynamic structure supporting extracellular matrix, secreting chemical messages (such as growth factors and cytokines), reacting to physical parameters (such as shear stress, tissue stiffness, and topography) and environmental signals (metabolites, hypoxia, etc.). With such versatile functions offered by the niche, there is no limit to its capacity of growth and replication.
- The AI runs are just a trial and error processes using brute force to cut open the ball of stem cells, have it re-shaped into specific form and left it to incubate. Eventually, they came upon an unstable configuration which could split relatively easy into two. Actually, it happened rarely and only in specific circumstances. This is probably what is referred to as the "new way of reproduction".

[End of 2022 Update]