PRP Injections – Platelet Rich Plasma Injections …

PRP injections are a new medical technology used in the treatment of musculoskeletal injuries as well as in the aesthetic medicine field. This website aims to provide information about this therapy and where it can be performed.

The field of sports medicine is advancing at a rapid pace and is encouraging millions of people from athletes to the general population to participate in regular activity to stay healthy and active (1). However this increase in physical activity has also seen an increase in musculoskeletal injuries. The World Health Organisation has recently recognised that musculoskeletal injuries affect hundreds of millions of people worldwide and are the most common cause of severe long term pain and physical disability (2). In Australia alone, there are almost 4 cases of medically treated sporting injuries per 100 persons (3).

Soft tissue injuries which include ligament and tendons represent up to 45% of all musculoskeletal injuries (4,5). The increasing participation in sporting activities coupled together with improved modern imaging techniques such as MRI and high resolution ultrasound have helped sports medicine practitioners to better understand these injuries (6).

References 1. Snchez M, Anitua E, Orive G, Mujika I, Andia I. Platelet-rich therapies in the treatment of orthopaedic sport injuries. Sports Med. 2009;39(5):345-54. 2. Woolf AD, Pfleyer B. Burdon of major musculoskeletal conditions. Bull World Health Organ. 2003;81:64656. 3. Cassel EP, Finch CF, Stathakis VZ. Epidemiology of medically treated sport and active recreation injuries in the Latrobe Valley, Victoria, Australia. Br J Sports Med 20032; 37: 405-9. 4. Anitua M, Snchez E, Nurden A, Nurden P, Orive G, And a I. New insights into and novel applications for platelet-rich fibrin therapies. Trends Biotechnol. 2006;24(5):22734. 5. Praemer AF. Musculoskeletal conditions in the United States. 2nd ed. Rosemont: American Academy of Orthopaedic Surgeons; 1999. 6. Sampson S, Gerhardt M, Mandelbaum B. Platelet rich plasma injection grafts for musculoskeletal injuries: a review. Curr Rev Musculoskelet Med. 2008 Dec;1(3-4):165-74.

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PRP Injections - Platelet Rich Plasma Injections ...

Embryonic stem cells controlled with light: Study reveals …

UC San Francisco researchers have for the first time developed a method to precisely control embryonic stem cell differentiation with beams of light, enabling them to be transformed into neurons in response to a precise external cue.

The technique also revealed an internal timer within stem cells that lets them tune out extraneous biological noise but transform rapidly into mature cells when they detect a consistent, appropriate molecular signal, the authors report in a study published online August 26 in Cell Systems.

"We've discovered a basic mechanism the cell uses to decide whether to pay attention to a developmental cue or to ignore it," said co-senior author Matthew Thomson, PhD, a researcher in the department of Cellular and Molecular Pharmacology and the Center for Systems and Synthetic Biology at UCSF.

During embryonic development, stem cells perform an elaborately timed dance as they transform from their neutral, undifferentiated form to construct all the major organ systems of the body. Researchers have identified many different molecular cues that signal stem cells when to transform into their mature form, whether it be brain or liver or muscle, at just the right time.

These discoveries have raised hopes that taking control of stem cells could let scientists repair damaged and aging tissues using the body's own potential for regeneration. But so far, getting stem cells to follow instructions en masse has proven far more difficult than researchers once expected.

In recent years, scientists have found that many of the genes encoding these developmental cues constantly flip on and off in undifferentiated stem cells. How the cells manage to ignore these noisy fluctuations but then respond quickly and decisively to authentic developmental cues has remained a mystery.

"These cells receive so many varied inputs," said lead author Cameron Sokolik, a Thomson laboratory research assistant at the time of the study. "The question is how does the cell decide when to differentiate?"

To test how stem cells interpret developmental cues as either crucial signals or mere noise, Thomson and colleagues engineered cultured mouse embryonic stem cells in which the researchers could use a pulse of blue light to switch on the Brn2 gene, a potent neural differentiation cue. By adjusting the strength and duration of the light pulses, the researchers could precisely control the Brn2 dosage and watch how the cells respond.

They discovered that if the Brn2 signal was strong enough and long enough, stem cells would quickly begin to transform into neurons. But if the signal was too weak or too brief, the cells ignored it completely.

"The cells are looking at the length of the signal," Thomson said. "That was a big surprise."

To learn how stem cells were able to weed out fleeting Brn2 signals but respond to persistent ones, co-senior author Stanley Qi, PhD, and co-author Yanxia Liu, PhD, both now at Stanford University, used the CRISPR-Cas9 gene editing system to add a fluorescent tag to the transcription factor Nanog, which normally acts as a brake on differentiation. This protein could then be used as a read-out on the cells' decision-making.

The team discovered that Nanog itself is actually key to the cells' impeccable sense of timing. When the Brn2 signal turns on, it disrupts a molecular feedback loop that keeps the cell stable and undifferentiated. In response, Nanog protein levels start to drop. However, the protein takes about four hours to dissipate completely, which makes Nanog an excellent internal stop-watch. If the Brn2 signal is a fluke, Nanog levels can quickly rebound and the cell will do nothing. On the other hand, if Nanog runs out and the Brn2 signal is still on, "it's like a buzzer goes off," Thomson said. "And once it goes, it really goes -- the cells rapidly start converting into neurons."

Thomson believes that similar timer mechanisms may govern stem cell differentiation into many different tissues.

"It's hard for a cell to be both tolerant and fast, to reject minor fluctuations, but respond very precisely and sharply when it sees a signal," he said. "This mechanism is able to do that."

Thomson is a UCSF Sandler Fellow and Systems Biology Fellow. Since 1998, these unique fellowship programs have enabled UCSF to recruit young researchers straight out of graduate school to pursue ambitious high-risk, high-reward science.

Thomson's ambitious big idea is to use the light-inducible differentiation technology his group has developed to study how stem cells produce complex tissues in three dimensions. He imagines a day when researchers can illuminate a bath of undifferentiated stem cells with a pattern of different colors of light and come back the next day to find a complex pattern of blood and nerve and liver tissue forming an organ that can be transplanted into a patient.

"There's lots of promise that we can do these miraculous things like tissue repair or even growing new organs, but in practice, manipulating stem cells has been notoriously noisy, inefficient, and difficult to control," Thomson said. "I think it's because the cell is not a puppet. It's an agent that is constantly interpreting information, like a brain. If we want to precisely manipulate cell fate, we have to understand the information-processing mechanisms in the cell that control how it responds to the things we're trying to do to it."

The rest is here:
Embryonic stem cells controlled with light: Study reveals ...

Researchers control embryonic stem cells with light

August 26, 2015 A colony of embryonic stem cells, from the H9 cell line (NIH code: WA09). Viewed at 10X with Carl Zeiss Axiovert scope. (The cells in the background are mouse fibroblast cells. Only the colony in the centre are human embryonic stem cells) Credit: Ryddragyn/ Wikipedia

UC San Francisco researchers have for the first time developed a method to precisely control embryonic stem cell differentiation with beams of light, enabling them to be transformed into neurons in response to a precise external cue.

The technique also revealed an internal timer within stem cells that lets them tune out extraneous biological noise but transform rapidly into mature cells when they detect a consistent, appropriate molecular signal, the authors report in a study published online August 26 in Cell Systems.

"We've discovered a basic mechanism the cell uses to decide whether to pay attention to a developmental cue or to ignore it," said co-senior author Matthew Thomson, PhD, a researcher in the department of Cellular and Molecular Pharmacology and the Center for Systems and Synthetic Biology at UCSF.

During embryonic development, stem cells perform an elaborately timed dance as they transform from their neutral, undifferentiated form to construct all the major organ systems of the body. Researchers have identified many different molecular cues that signal stem cells when to transform into their mature form, whether it be brain or liver or muscle, at just the right time.

These discoveries have raised hopes that taking control of stem cells could let scientists repair damaged and aging tissues using the body's own potential for regeneration. But so far, getting stem cells to follow instructions en masse has proven far more difficult than researchers once expected.

In recent years, scientists have found that many of the genes encoding these developmental cues constantly flip on and off in undifferentiated stem cells. How the cells manage to ignore these noisy fluctuations but then respond quickly and decisively to authentic developmental cues has remained a mystery.

"These cells receive so many varied inputs," said lead author Cameron Sokolik, a Thomson laboratory research assistant at the time of the study. "The question is how does the cell decide when to differentiate?"

To test how stem cells interpret developmental cues as either crucial signals or mere noise, Thomson and colleagues engineered cultured mouse embryonic stem cells in which the researchers could use a pulse of blue light to switch on the Brn2 gene, a potent neural differentiation cue. By adjusting the strength and duration of the light pulses, the researchers could precisely control the Brn2 dosage and watch how the cells respond.

They discovered that if the Brn2 signal was strong enough and long enough, stem cells would quickly begin to transform into neurons. But if the signal was too weak or too brief, the cells ignored it completely.

"The cells are looking at the length of the signal," Thomson said. "That was a big surprise."

To learn how stem cells were able to weed out fleeting Brn2 signals but respond to persistent ones, co-senior author Stanley Qi, PhD, and co-author Yanxia Liu, PhD, both now at Stanford University, used the CRISPR-Cas9 gene editing system to add a fluorescent tag to the transcription factor Nanog, which normally acts as a brake on differentiation. This protein could then be used as a read-out on the cells' decision-making.

The team discovered that Nanog itself is actually key to the cells' impeccable sense of timing. When the Brn2 signal turns on, it disrupts a molecular feedback loop that keeps the cell stable and undifferentiated. In response, Nanog protein levels start to drop. However, the protein takes about four hours to dissipate completely, which makes Nanog an excellent internal stop-watch. If the Brn2 signal is a fluke, Nanog levels can quickly rebound and the cell will do nothing. On the other hand, if Nanog runs out and the Brn2 signal is still on, "it's like a buzzer goes off," Thomson said. "And once it goes, it really goes - the cells rapidly start converting into neurons."

Thomson believes that similar timer mechanisms may govern stem cell differentiation into many different tissues.

"It's hard for a cell to be both tolerant and fast, to reject minor fluctuations, but respond very precisely and sharply when it sees a signal," he said. "This mechanism is able to do that."

Thomson is a UCSF Sandler Fellow and Systems Biology Fellow. Since 1998, these unique fellowship programs have enabled UCSF to recruit young researchers straight out of graduate school to pursue ambitious high-risk, high-reward science.

Thomson's ambitious big idea is to use the light-inducible differentiation technology his group has developed to study how stem cells produce complex tissues in three dimensions. He imagines a day when researchers can illuminate a bath of undifferentiated stem cells with a pattern of different colors of light and come back the next day to find a complex pattern of blood and nerve and liver tissue forming an organ that can be transplanted into a patient.

"There's lots of promise that we can do these miraculous things like tissue repair or even growing new organs, but in practice, manipulating stem cells has been notoriously noisy, inefficient, and difficult to control," Thomson said. "I think it's because the cell is not a puppet. It's an agent that is constantly interpreting information, like a brain. If we want to precisely manipulate cell fate, we have to understand the information-processing mechanisms in the cell that control how it responds to the things we're trying to do to it."

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Researchers control embryonic stem cells with light

Glutamate and glutamate receptors in the vertebrate retina …

Victoria Connaughton

1. General overview of synaptic transmission.

Cells communicate with each other electrically, through gap junctions, and chemically, using neurotransmitters. Chemical synaptic transmission allows nerve signals to be exchanged between cells which are electrically isolated from each other. The chemical messenger, or neurotransmitter, provides a way to send the signal across the extracellular space, from the presynaptic neuron to the postsynaptic cell. The space is called acleft and is typically more than 10 nanometers across. Neurotransmitters are synthesized in the presynaptic cell and stored in vesicles in presynaptic processes, such as the axon terminal. When the presynaptic neuron is stimulated, calcium channels open and the influx of calcium ions into the axon terminal triggers a cascade of events leading to the release of neurotransmitter. Once released, the neurotransmitter diffuses across the cleft and binds to receptors on the postsynaptic cell, allowing the signal to propagate. Neurotransmitter molecules can also bind onto presynaptic autoreceptors and transporters, regulating subsequent release and clearing excess neurotransmitter from the cleft. Compounds classified as neurotransmitters have several characteristics in common (reviewed in Massey, 1990, Erulkar, 1994). Briefly, (1) the neurotransmitter is synthesized, stored, and released from the presynaptic terminal. (2) Specific neurotransmitter receptors are localized on the postsynaptic cells, and (3) there exists a mechanism to stop neurotransmitter release and clear molecules from the cleft. Common neurotransmitters in the retina are glutamate, GABA, glycine, dopamine, and acetylcholine. Neurotransmitter compounds can be small molecules, such as glutamate and glycine, or large peptides, such as vasoactive intestinal peptide (VIP). Some neuroactive compounds are amino acids, which also have metabolic functions in the presynaptic cell.

Fig. 1. Structure of the glutamate molecule

Glutamate (Fig. 1) is believed to be the major excitatory neurotransmitter in the retina. In general, glutamate is synthesized from ammonium and alpha-ketoglutarate (a component of the Krebs Cycle) and is used in the synthesis of proteins, other amino acids, and even other neurotransmitters (such as GABA; Stryer, 1988). Though glutamate is present in all neurons, only a few are glutamatergic, releasing glutamate as their neurotransmitter. Neuroactive glutamate is stored in synaptic vesicles in presynaptic axon terminals (Fykse and Fonnum, 1996). Glutamate is incorporated into the vesicles by a glutamate transporter located in the vesicular membrane. This transporter selectively accumulates glutamate through a sodium-independent, ATP-dependent process (Naito and Ueda, 1983, Tabb and Ueda, 1991, Fykse and Fonnum, 1996), resulting in a high concentration of glutamate in each vesicle. Neuroactive glutamate is classified as an excitatory amino acid (EAA) because glutamate binding onto postsynaptic receptors typically stimulates, or depolarizes, the postsynaptic cells.

2. Histological techniques identify glutamatergic neurons.

Fig. 2. Glutamate immunoreactivity

Using immunocytochemical techniques, neurons containing glutamate are identified and labeled with a glutamate antibody. In the retina, photoreceptors, bipolar cells, and ganglion cells are glutamate immunoreactive (Ehinger et al, 1988, Marc et al., 1990, Van Haesendonck and Missotten, 1990, Kalloniatis and Fletcher, 1993, Yang and Yazulla, 1994, Jojich and Pourcho, 1996) (Fig. 2). Some horizontal and/or amacrine cells can also display weak labeling with glutamate antibodies (Ehinger et al., 1988, Marc et al., 1990, Jojich and Pourcho, 1996; Yang, 1996). These neurons are believed to release GABA, not glutamate, as their neurotransmitter (Yazulla, 1986), suggesting the weak glutamate labeling reflects the pool of metabolic glutamate used in the synthesis of GABA. This has been supported by the results from double-labeling studies using antibodies to both GABA and glutamate: glutamate-positive amacrine cells also label with the GABA antibodies (Jojich and Pourcho, 1996, Yang, 1996).

Fig. 3. Autoradiogram of glutamate uptake through glutamate transporters

Photoreceptors, which contain glutamate, actively take up radiolabeled glutamate from the extracellular space, as do Muller cells (Fig. 3) (Marc and Lam, 1981; Yang and Wu, 1997). Glutamate is incorporated into these cell types through a high affinity glutamate transporter located in the plasma membrane. Glutamate transporters maintain the concentration of glutamate within the synaptic cleft at low levels, preventing glutamate-induced cell death (Kanai et al., 1994). Though Muller cells take up glutamate, they do not label with glutamate antibodies (Jojich and Pourcho, 1996). Glutamate incorporated into Muller cells is rapidly broken down into glutamine, which is then exported from glial cells and incorporated into surrounding neurons (Pow and Crook, 1996). Neurons can then synthesize glutamate from glutamine (Hertz, 1979, Pow and Crook, 1996).

Thus, histological techniques are used to identify potential glutamatergic neurons by labeling neurons containing glutamate (through immunocytochemistry) and neurons that take up glutamate (through autoradiography). To determine if these cell types actually release glutamate as their neurotransmitter, however, the receptors on postsynaptic cells have to be examined.

3. Glutamate receptors.

Once released from the presynaptic terminal, glutamate diffuses across the cleft and binds onto receptors located on the dendrites of the postsynaptic cell(s). Multiple glutamate receptor types have been identified. Though glutamate will bind onto all glutamate receptors, each receptor is characterized by its sensitivity to specific glutamate analogues and by the features of the glutamate-elicited current. Glutamate receptor agonists and antagonists are structurally similar to glutamate (Fig. 4), which allows them to bind onto glutamate receptors. These compounds are highly specific and, even in intact tissue, can be used in very low concentrations because they are poor substrates for glutamate uptake systems (Tachibana and Kaneko, 1988, Schwartz and Tachibana, 1990).

Fig. 4. Glutamate receptor agonists and antagonists

Two classes of glutamate receptors (Fig. 5) have been identified: (1) ionotropic glutamate receptors, which directly gate ion channels, and (2) metabotropic glutamate receptors, which may be coupled to an ion channel or other cellular functions via an intracellular second messenger cascade. These receptor types are similar in that they both bind glutamate and glutamate binding can influence the permeability of ion channels. However, there are several differences between the two classes.

Fig. 5. Ionotropic and metabotropic glutamate receptors and channels

4. Ionotropic glutamate receptors.

Glutamate binding onto an ionotropic receptor directly influences ion channel activity because the receptor and the ion channel form one complex (Fig. 5a). These receptors mediate fast synaptic transmission between neurons. Each ionotropic glutamate receptor, or iGluR, is formed from the co-assembly of individual subunits. The assembled subunits may or may not be homologous, with the different combinations of subunits resulting in channels with different characteristics (Keinanen et al., 1990, Verdoorn et al., 1991, Moyner et al., 1992; Nakanishi, 1992, Ozawa and Rossier, 1996).

Fig. 6. Comparison between NMDA and non-NMDA receptors

Two iGluR types (see Fig. 6) have been identified: (1) NMDA receptors, which bind glutamate and the glutamate analogue N-Methyl-D-Aspartate (NMDA) and (2) non-NMDA receptors, which are selectively agonized by kainate, AMPA, and quisqualate, but not NMDA.

Non-NMDA receptors. Glutamate binding onto a non-NMDA receptor opens non-selective cation channels more permeable to sodium (Na+) and potassium (K+) ions than calcium (Ca+2) (Mayer and Westbrook, 1987). Glutamate binding elicits a rapidly activating inward current at membrane potentials negative to 0 mV, and an outward current at potentials positive to 0 mV. Kainate, quisqualate, and AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) are the specific agonists at these receptors; CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dione-benzo[f]quinoxaline-7-sulfonamide), and DNQX (6,7-dinitroquinoxaline-2,3-dione) are the antagonists.

Fig. 7. Whole-cell patch clamp to show quisqualate and kainate gated currents

In retina, non-NMDA receptors have been identified on horizontal cells, OFF-bipolar cells, amacrine cells, and ganglion cells (see below). Patch clamp recordings (Gilbertson et al., 1991, Zhou et al., 1993, Boos et al., 1993, Cohen and Miller, 1994, Yu and Miller, 1995) indicate that AMPA, quisqualate, and/or kainate application can evoke currents in these cells. However, the kinetics of the ligand-gated currents differ. AMPA and quisqualate-elicited currents rapidly desensitize; whereas, kainate-gated currents do not (Fig. 7a). The desensitization at AMPA/quisqualate receptors can be reduced (Fig. 7b) by adding cyclothiazide (Yamada and Tang, 1993), which stabilizes the receptor in an active (or non-desensitized) state (Yamada and Tang, 1993, Kessler et al., 1996).

Each non-NMDA receptor is formed from the co-assembly of several subunits (Boulter et al., 1990, Nakanishi et al., 1990, Nakanishi, 1992). To date, seven subunits (named GluR1 through GluR7) have been cloned (Hollmann et al., 1989, Boulter et al., 1990, Keinanen et al., 1990, Nakanishi et al., 1990, Bettler et al., 1990, 1992, Egebjerg et al., 1991). Expression of subunit clones in Xenopus oocytes revealed that GluR5, GluR6, and GluR7 (along with subunits KA1 and KA2) co-assemble to form kainate(-preferring) receptors; whereas, GluR1, GluR2, GluR3, and GluR4 are assembled into AMPA(-preferring) receptors (Nakanishi, 1992).

NMDA receptors. Glutamate binding onto an NMDA receptor also opens non-selective cation channels, resulting in a conductance increase. However, the high conductance channel associated with these receptors is more permeable to Ca+2 than Na+ ions (Mayer and Westbrook, 1987) and NMDA-gated currents typically have slower kinetics than kainate- and AMPA-gated channels. As the name suggests, NMDA is the selective agonist at these receptors. The compounds MK-801, AP-5 (2-amino-5-phosphonopentanoic acid), and AP-7 (2-amino-7-phosphoheptanoic acid) are NMDA receptor antagonists.

NMDA receptors are structurally complex, with separate binding sites for glutamate, glycine, magnesium ions (Mg+2), zinc ions (Zn+2), and a polyamine recognition site (Fig. 6b). There is also an antagonist binding site for PCP and MK-801 (Lodge, 1997). The glutamate, glycine, and magnesium binding sites are important for receptor activation and gating of the ion channel. In contrast, the zinc and polyamine sites are not needed for receptor activation, but affect the efficacy of the channel. Zinc blocks the channel in a voltage-independent manner (Westbrook and Mayer, 1987). The polyamine site (Ransom and Stec, 1988, Williams et al., 1994) binds compounds such as spermine or spermidine, either potentiating (Ranson and Stec, 1988; Williams et al., 1994) or inhibiting (Williams et al., 1994) the activity of the receptor, depending on the combination of subunits forming each NMDA receptor (Williams et al., 1994).

To date, five subunits (NR1, NR2a, N2b, N2c, and N2d) of NMDA receptors have been cloned (Moriyoshi et al., 1991, Ikeda et al., 1992, Katsuwada et al., 1992, Meguro et al., 1992, Ishii et al., 1993). As with non-NMDA receptors, NMDA receptor subunits can co-assemble as homomers (i.e., five NR1 subunits; Moyner et al., 1992, Moriyoshi et al., 1992) or heteromers (one NR1 + four NR2 subunits; Meguro et al., 1992, Katsuwada et al., 1992, Moyner et al., 1992, Ishii et al., 1993). However, all functional NMDA receptors express the NR1 subunit (Moyner et al., 1992, Nakanishi, 1992, Ishii et al., 1993).

Fig. 8. NMDA receptor activation

The glutamate, glycine, and Mg+2 binding sites confer both ligand-gated and voltage-gated properties onto NMDA receptors. NMDA receptors are ligand-gated because the binding of glutamate (ligand) is required to activate the channel. In addition, micromolar concentrations of glycine must also be present (Fig. 8) (Johnson and Ascher, 1987, Kleckner and Dingledine, 1988). The requirement for both glutamate and glycine makes them co-agonists (Kleckner and Dingledine, 1988) at NMDA receptors.

Mg+2 ions provide a voltage-dependent block of NMDA-gated channels (Nowak et al., 1984). This can be seen in the current-voltage (I-V) relationship presented in Fig. 9 (from Nowak et al., 1984).

Fig. 9. Mg+2 ions block NMDA receptor channels

I-V curves plotted from currents recorded in the presence of Mg+2 have a characteristic J-shape (dotted line); whereas, a linear relationship is calculated in Mg+2-free solutions (solid line). At negative membrane potentials, Mg+2 ions occupy the binding site causing less current to flow through the channel. As the membrane depolarizes, the Mg+2 block is removed (Nowak et al., 1984).

Retinal ganglion cells and some amacrine cell types express functional NMDA receptors in addition to non-NMDA receptors (i.e., Massey and Miller, 1988, 1990, Mittman et al., 1990, Dixon and Copenhagen 1992, Diamond and Copenhagen, 1993, Cohen and Miller, 1994). The currents elicited through these different iGluR types can be distinguished pharmacologically. Non-NMDA receptor antagonists block a transient component of the ganglion cell light response; whereas, NMDA receptor antagonists block a more sustained component (Mittman et al., 1990, Diamond and Copenhagen, 1993, Hensley et al., 1993, Cohen and Miller, 1994). These findings suggest the currents elicited through co-localized NMDA and non-NMDA receptors mediate differential contributions to the ON- and OFF-light responses observed in ganglion cells (i.e., Diamond and Copenhagen, 1993).

5. Metabotropic glutamate receptors.

Unlike ionotropic receptors, which are directly linked to an ion channel, metabotropic receptors are coupled to their associated ion channel through a second messenger pathway. Ligand (glutamate) binding activates a G-protein and initiates an intracellular cascade (Nestler and Duman, 1994). Metabotropic glutamate receptors (mGluRs) are not co-assembled from multiple subunits, but are one polypeptide (Fig. 5b). To date, eight mGluRs (mGluR1-mGluR8) have been cloned (Houamed et al., 1991, Masu et al., 1991, Abe et al., 1992, Tanabe et al., 1992, Nakajima et al., 1993, Saugstad et al., 1994, Duvoisin et al., 1995). These receptors are classified into three groups (I, II, and III) based on structural homology, agonist selectivity, and their associated second messenger cascade (Table 1, end of chapter) (reviewed in Nakanishi, 1994, Knopfel et al., 1995, Pin and Duvoisin, 1995, Pin and Bockaert, 1995).

In brief, Group I mGluRs (mGluR1 and mGluR5) are coupled to the hydrolysis of fatty acids and the release of calcium from internal stores. Quisqualate and trans-ACPD are Group I agonists. Group II (mGluR2 and mGluR3) and Group III (mGluRs 4, 6, 7, and 8) receptors are considered inhibitory because they are coupled to the downregulation of cyclic nucleotide synthesis (Pin and Duvoisin, 1995). L-CCG-1 and trans-ACPD agonize Group II receptors; L-AP4 (also called APB) selectively agonizes Group III receptors. In situ hybridization studies have revealed that the mRNAs encoding Group I, II, and III mGluRs are present in retina (see below); however, with the exception of the APB receptor, the function of all these receptor types in retina has not been characterized.

APB receptor. In contrast to non-NMDA and NMDA receptors, glutamate binding onto an APB receptor elicits a conductance decrease (Slaughter and Miller, 1981, Nawy and Copenhagen, 1987, 1990) due to the closure of cGMP-gated non-selective cation channels (Nawy and Jahr, 1990) (Fig. 10).

Fig. 10.Whole-cell current traces to show kinetics of APB receptor gated currents

APB application selectively blocks the ON-pathway in the retina (Fig. 11) (Slaughter and Miller, 1981), i.e., ON-bipolar cell responses and the ON-responses in amacrine cells (Taylor and Wassle, 1995) and ganglion cells (Cohen and Miller, 1994, Kittila and Massey, 1995, Jin and Brunken, 1996) are eliminated by APB. Experimental evidence (Slaughter and Miller 1981, Massey et al., 1983) suggests the APB receptor is localized to ON-bipolar cell dendrites. Inhibition of amacrine and ganglion cell light responses, therefore, is due to a decrease in the input from ON-bipolar cells, not a direct effect on postsynaptic receptors.

Fig. 11. Intracellular recordings to show that APB selectively antagonizes the ON-pathways

APB (2-amino-4-phosphobutyric acid, also called L-AP4) is the selective agonist for all Group III mGluRs (mGluR4, 6, 7, and 8). So, which is the APB receptor located on ON-bipolar cell dendrites? MGluR4, 7, and 8 expression has been observed in both the inner nuclear layer and the ganglion cell layer (Duvoisin et al., 1995, Hartveit et al., 1995) suggesting these mGluRs are associated with more than one cell type. In contrast, mGluR6 expression has been localized to the INL (Nakajima et al., 1993, Hartveit et al., 1995) and the OPL (Nomura et al., 1994) where bipolar cell somata and dendrites are located. Furthermore, ON-responses are abolished in mice lacking mGluR6 expression (Masu et al., 1995). These mutants also display abnormal ERG b-waves, suggesting an inhibition of the ON-retinal pathway at the level of bipolar cells (Masu et al., 1995). Taken together, these findings suggest the APB receptor on ON-bipolar cells is mGluR6.

6. Glutamate transporters and transporter-like receptors.

Glutamate transporters have been identified on photoreceptors (Marc and Lam, 1981, Tachibana and Kaneko, 1988, Eliasof and Werblin, 1993) and Muller cells (Marc and Lam, 1981, Yang and Wu, 1997). From glutamate labeling studies, the average concentration of glutamate in photoreceptors, bipolar cells, and ganglion cells is 5mM (Marc et al. 1990). Physiological studies using isolated cells indicate that only M levels of glutamate are required to activate glutamate receptors (i.e., Aizenman et al., 1988, Zhou et al., 1993, Sasaki and Kaneko, 1996). Thus, the amount of glutamate released into the synaptic cleft is several orders of magnitude higher than the concentration required to activate most postsynaptic receptors. High affinity glutamate transporters located on adjacent neurons and surrounding glial cells rapidly remove glutamate from the synaptic cleft to prevent cell death (Kanai et al., 1994). Five glutamate transporters, EAAT-1 (or GLAST), EAAT-2 (or GLT-1), EAAT-3 (or EAAC-1), EAAT-4, and EAAT-5, have been cloned (Kanai and Hediger, 1992, Pines et al., 1992, Fairman et al., 1995, Schultz and Stell, 1996, Arriza et al., 1997, Kanai et al., 1997).

Glutamate transporters are pharmacologically distinct from both iGluRs and mGluRs. L-glutamate, L-aspartate, and D-aspartate are substrates for the transporters (Brew and Attwell, 1987, Tachibana and Kaneko, 1988, Eliasof and Werblin, 1993); glutamate receptor agonists (Brew and Attwell, 1987, Tachibana and Kaneko, 1988, Schwartz and Tachibana, 1990, Eliasof and Werblin, 1993) and antagonists (Barbour et al., 1991, Eliasof and Werblin, 1993) are not. Glutamate uptake can be blocked by the transporter blockers dihydrokainate (DHKA) and DL-threo-beta-hydroxyaspartate (HA) (Barbour et al., 1991, Eliasof and Werblin 1993).

Fig. 12 Glutamate transporters in Muller cells are electrogenic

Glutamate transporters incorporate glutamate into Muller cells along with the co-transport of three Na+ ions (Brew and Attwell, 1987, Barbour et al., 1988) and the antiport of one K+ ion (Barbour et al., 1988, Bouvier et al., 1992) and either one OH- or one HCO3- ion (Bouvier et al., 1992) (Fig. 12). The excess sodium ions generate a net positive inward current which drives the transporter (Brew and Attwell, 1987, Barbour et al., 1988). More recent findings indicate a glutamate-elicited chloride current is also associated with some transporters (Eliasof and Jahr, 1996, Arriza et al., 1997).

It should be noted that the glutamate transporters located in the plasma membrane of neuronal and glial cells (discussed in this section) are different from the glutamate transporters located on synaptic vesicles within presynaptic terminals (see section 1). The transporters in the plasma membrane transport glutamate in a Na+- and voltage-dependent manner independent of chloride (Brew and Attwell, 1987, Barbour et al., 1988, Kanai et al., 1994). L-glutamate, L-aspartate, and D-aspartate are substrates for these transporters (i.e., Brew and Attwell, 1987). In contrast, the vesicular transporter selectively concentrates glutamate into synaptic vesicles in a Na+-independent, ATP-dependent manner (Naito and Ueda, 1983, Tabb and Ueda, 1991, Fykse and Fonnum, 1996) that requires chloride (Tabb and Ueda, 1991, Fykse and Fonnum, 1996).

Glutamate receptors with transporter-like pharmacology have been described in photoreceptors (Picaud et al., 1995a, b, Grant and Werblin, 1996) and ON-bipolar cells (Grant and Dowling 1995, 1996). These receptors are coupled to a chloride current. The pharmacology of these receptors is similar to that described for glutamate transporters, as the glutamate-elicited current is (1) dependent upon external Na+, (2) reduced by transporter blockers, and (3) insensitive to glutamate agonists and antagonists. However, altering internal Na+ concentration does not change the reversal potential (Picaud et al., 1995b) or the amplitude (Grant and Werblin, 1995, Grant and Dowling, 1996) of the glutamate-elicited current, suggesting the receptor is distinct from glutamate transporters. At the photoreceptor terminals, the glutamate-elicited chloride current may regulate membrane potential and subsequent voltage-gated channel activity (i.e., Picaud et al., 1995a). Postsynaptically, this receptor is believed to mediate conductance changes underlying photoreceptor input to ON- cone bipolar cells (Grant and Dowling, 1995).

7. Localization of glutamate receptor types in the retina.

Fig. 13. The types of neurons in the vertebrate retina

Photoreceptor, bipolar, ganglion cells comprise the vertical transduction pathway in the retina. This pathway is modulated by lateral inputs from horizontal cells in the distal retina and amacrine cells in the proximal retina (Fig. 13). As described in the previous sections, photoreceptor, bipolar, and ganglion cells show glutamate immunoreactivity. Glutamate responses have been electrically characterized in horizontal and bipolar cells, which are postsynaptic to photoreceptors, and in amacrine and ganglion cells, which are postsynaptic to bipolar cells. Taken together, these results suggest glutamate is the neurotransmitter released by neurons in the vertical pathway. Recent in situ hybridization and immunocytochemical studies have localized the expression of iGluR subunits, mGluRs, and glutamate transporter proteins in the retina. These findings are summarized below.

8. Retinal neurons expressing ionotropic glutamate receptors.

In both higher and lower vertebrates, electrophysiological recording techniques have identified ionotropic glutamate receptors on the neurons comprising the OFF-pathway (Table 2, end of chapter). In the distal retina, OFF-bipolar cells (Fig. 14) (Euler et al., 1996, Sasaki and Kaneko, 1996, Hartveit, 1997) and horizontal cells (Fig. 15) (Yang and Wu, 1991, Zhou et al., 1993, Kriaj et al., 1994) respond to kainate, AMPA, and quisqualate application, but not NMDA nor APB. (However, NMDA receptors have been identified on catfish horizontal cells (ODell and Christensen, 1989, Eliasof and Jahr, 1997) and APB-induced hyperpolarizations have been reported in some fish horizontal cells (Nawy et al., 1989, Takahashi and Copenhagen, 1992, Furukawa et al., 1997)).

Non-NMDA agonists also stimulate both amacrine cells (Fig. 16a) (Massey and Miller, 1988, Dixon and Copenhagen, 1992, Boos et al., 1993) and ganglion cells (Fig. 16b) (Mittman et al., 1990, Diamond and Copenhagen, 1993, Hensley et al., 1993, Cohen and Miller, 1994, Yu and Miller, 1995). Ganglion cells responses to NMDA have been observed (Massey and Miller, 1988, 1990, Mittman et al., 1990, Diamond and Copenhagen, 1993, Cohen and Miller, 1994); whereas, NMDA responses have been recorded in only some types of amacrine cells (Massey and Miller, 1988, Dixon and Copenhagen, 1992, Boos et al., 1993, but see Hartveit and Veruki, 1997).

Fig. 16. Glutamate receptors on amacrine and ganglion cells

Consistent with this physiological data, antibodies to the different non-NMDA receptor subunits differentially label all retinal layers (Table 3, end of chapter; Hartveit et al., 1994, Peng et al., 1995, Hughes, 1997, Pourcho et al., 1997) and mRNAs encoding the different non-NMDA iGluR subunits are similarly expressed (Hughes et al., 1992, Hamassaki-Britto et al., 1993, Brandstatter et al., 1994). In contrast, mRNAs encoding NMDA subunits are expressed predominantly in the proximal retina, where amacrine and ganglion cells are located (INL, IPL, GCL; Table 3) (Brandstatter et al., 1994, Hartveit et al., 1994), though mRNA encoding the NR2a subunit (Hartveit et al., 1994) has been observed in the OPL and antibodies to the NR2d (Wenzel et al., 1997) and the NR1 subunits (Hughes, 1997) label rod bipolar cells.

9. Retinal neurons expressing metabotropic glutamate receptors.

All metabotropic glutamate receptors, except mGluR3, have been identified in retina either through antibody staining (Peng et al., 1995, Brandstatter et al., 1996, Koulen et al., 1997, Pourcho et al., 1997) or in situ hybridization (Nakajima et al., 1993, Duvoisin et al., 1995, Hartveit et al., 1995). MGluRs are differentially expressed throughout the retina, specifically in the outer plexiform layer, inner nuclear layer, inner plexiform layer, and the ganglion cell layer (Table 4, end of chapter). Though different patterns of mGluR expression have been observed in the retina, only the APB receptor on ON-bipolar cells has been physiologically examined.

10. Retinal neurons expressing glutamate transporters.

The glutamate transporters GLAST, EAAC1, and GLT-1have been identified in retina (Table 5, end of chapter). GLAST (L-glutamate/L-aspartatetransporter) immunoreactivity is found in all retinal layers (Otori et al. 1994), but not in neuronal tissue. GLAST is localized to Muller cell membranes (Otori et al. 1994, Derouiche and Rauen, 1995, Rauen et al., 1996, Lehre et al., 1997). In contrast, EAAC-1 (excitatoryaminoacidcarrier-1) antibodies do not label Muller cells or photoreceptors. EAAC-1 immunoreactivity is observed in ganglion and amacrine cells in chicken, rat, goldfish, and turtle retinas. In addition, bipolar cells positive labeled with EAAC-1 antibody in lower vertebrates and immunopositive horizontal cells were observed in rat (Schultz and Stell, 1996). GLT-1 (glutamatetransporter-1) proteins have been identified in monkey (Grunert et al., 1994), rat (Rauen et al., 1996), and rabbit (Massey et al., 1997) bipolar cells. In addition, a few amacrine cells were weakly labeled with the GLT-1 antibody in rat (Rauen et al., 1996), as were photoreceptor terminals in rabbit (Massey et al., 1997).

11. Summary and conclusions.

Fig. 17. The ribbon glutamatergic synapse in the retina

Histological analyses of presynaptic neurons and physiological recordings from postsynaptic cells suggest photoreceptor, bipolar, and ganglion cells release glutamate as their neurotransmitter. Multiple glutamate receptor types are present in the retina. These receptors are pharmacologically distinct and differentially distributed. IGluRs directly gate ion channels and mediate rapid synaptic transmission through either kainate/AMPA or NMDA receptors. Glutamate binding onto iGluRs opens cation channels, depolarizing the postsynaptic cell membrane. Neurons within the OFF-pathway (horizontal cells, OFF-bipolar cells, amacrine cells, and ganglion cells) express functional iGluRs. MGluRs are coupled to G-proteins. Glutamate binding onto mGluRs can have a variety of effects depending on the second messenger cascade to which the receptor is coupled. The APB receptor, found on ON-bipolar cell dendrites, is coupled to the synthesis of cGMP. At these receptors, glutamate decreases cGMP formation leading to the closure of ion channels. Glutamate transporters, found on glial and photoreceptor cells, are also present at glutamatergic synapses (Fig. 17). Transporters remove excess glutamate from the synaptic cleft to prevent neurotoxicity. Thus, postsynaptic responses to glutamate are determined by the distribution of receptors and transporters at a glutamatergic synapses which, in retina, determine the conductance mechanisms underlying visual information processing within the ON- and OFF-pathways.

Tables

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Glutamate and glutamate receptors in the vertebrate retina ...

STEMCELL Technologies Inc. Enters a Licensing Agreement …

VANCOUVER, British Columbia--(BUSINESS WIRE)--STEMCELL Technologies Inc. has signed a patent license agreement with iPS Academia Japan, Inc. to license and commercialize iPS Academia Japan, Inc.s patented technologies for induced pluripotent stem cell (iPS cell) research.

This agreement will enable STEMCELL Technologies to develop, manufacture, and distribute products that are optimized for cellular reprogramming. This further expands STEMCELLs extensive portfolio of products for hPSC research, which includes ReproTeSR and TeSR-E7 media for reprogramming, the TeSR family of maintenance media, and the STEMdiff product line for robust and reliable differentiation to various cell lineages.

The patents related to iPS cell technologies licensed by iPS Academia Japan are the result of the groundbreaking research of Professor Shinya Yamanaka of the Center for iPS Cell Research and Application (CiRA) at Kyoto University, Japan. iPS cells hold immense potential for drug development and disease modeling. Somatic cells can be reprogrammed using iPS cell technology and subsequently differentiated into specific cell types of diverse lineages. This enables researchers to develop cell lines for, among other applications, screening potential treatments at the patient level. The personalized nature of this approach ensures greater predictive accuracy in disease modeling and treatment outcome.

About STEMCELL Technologies Inc. As Scientists Helping Scientists, STEMCELL Technologies is committed to providing high-quality cell culture media, cell isolation products and accessory reagents for life science research. Driven by science and a passion for quality, STEMCELL Technologies provides over 2000 products to more than 70 countries worldwide. Our specialty cell culture reagents, instruments and tools are designed to support science along the basic to translational research continuum. To learn more, visit http://www.stemcell.com.

About iPS Academia Japan, Inc. iPS Academia Japan, Inc. (AJ) is an affiliate of Kyoto University, and its main role is to manage and utilize the patents and other intellectual properties held/controlled by Kyoto University and other institutions in the field of iPS cell technologies so that the research results contribute to health and welfare worldwide. AJ was established in Kyoto in June 2008. AJ's patent portfolio consists of approximately 110 patent families (350 patent application cases) in iPS cell technologies as of July 2015, and approximately 150 license arrangements have been executed with domestic or international entities.For more information, visit http://www.ips-cell.net.

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STEMCELL Technologies Inc. Enters a Licensing Agreement ...

Home Page of the Human Gene and Cell Therapy Center

Although there appears to be no definitive cure for many human genetic diseases, there are some gene therapy products awaiting for FDA approval and expected to be released in the near future. So far, more than 2,000 clinical gene therapy trials were conducted worldwide involving tens of thousands of patients. Some of the gene therapy drugs are commercialized and already in the market. Consequently, there is a dramatic progress made in the clinical outcome of current gene therapy trials such as those conducted against Leber's Congenital Amaurosis (LCA), Thalassemia, SCID, Hemophilia and Muscular Dystrophy etc.

In October 2012, the European Commission granted marketing authorization for Glybera (alipogene tiparvovec) under exceptional circumstances as a treatment for adult patients diagnosed with familial lipoprotein lipase deficiency (LPLD) confirmed by genetic testing, and suffering from severe or multiple pancreatitis attacks despite dietary fat restrictions. Glybera is a gene therapy drug that is designed to restore the LPL enzyme activity required to enable the processing, or clearance, of fat-carrying chylomicron particles formed in the intestine after a fat-containing meal. The product consists of an engineered copy of the human LPL gene packaged with a tissue-specific promoter in a non-replicating AAV1 vector, which has a particular affinity for muscle cells.

Human Gene and Cell Therapy Center of Akdeniz University Hospitals and Clinics was initially established as the Human Gene Therapy Unit of College of Medicine by Dr. Salih Sanlioglu in 2003. In 2010, the unit was restructured as a center to include cell therapy studies as well. The Gene and Cell Therapy Center is currently located on the first floor of the Institute for Transplantation, Akdeniz University Hospitals and Clinics. Since clinical grade vector production requires cGMP laboratories, the Center has been scheduled to be relocated to a new R&D building in the near future.

Although, initial studies concerned the development of novel gene therapy methods for various cancer types such as cancers of the lung, prostate and breast etc. including auto-immune diseases like rheumatoid arthritis, gene and cell therapy of diabetes became the priority in research due to alarming incidence of diabetes in the whole world. Accordingly, pancreatic islet transplantation fortified with gene delivery became a popular area of interest in the Center. These scientific projects relating to the diabetes treatment necessitated international collaboration to complete.

Some of the gene delivery vectors constructed and produced in the lab are shown on the right. These gene delivery vehicles are the most advanced gene therapy vectors normally used in gene therapy clinical trials. Adenovirus vectors are prefered in cancer gene therapy applications due to their antigenicity while AAV or Lentivirus vector is chosen when long term gene expression is desired.

For an instance, Alipogene tiparvovec (marketed under the trade name Glybera) is a AAV based gene therapy treatment designed to compensate for lipoprotein lipase deficiency (LPLD). It is the first of its kind to be approved as a gene therapy vector in the western world.

Nonetheless, due to outstanding quality of research and the scientific accomplishments, the center stands out to be one of the pioneering gene and cell therapy research facilities in the country. Accordingly, experimental gene and cell therapy protocols developed in the Center are published in the leading gene therapy journals like Human Gene Therapy, Cancer Gene Therapy, and Current Gene Therapy and diabetes journals etc. The founder of the Gene and Cell Therapy Center and his research activities are outlined below:

After graduating from the School of Veterinary Medicine, Selcuk University, Turkiye, Dr. Salih Sanlioglu attended the Ohio State University College of Medicine, where he obtained his Master's (1992) and PhD (1996) degrees in the field of Molecular Genetics.

Following completion of his postdoctoral training at the Human Gene Therapy Institute of University of Pennsylvania (1997) and internal medicine research fellowship at the Gene Therapy Center of University of Iowa (2002), Dr. Sanlioglu has joined the medical faculty of Akdeniz University, Antalya, Turkiye, and subsequently established the first human gene therapy facility in the country.

Although, his initial studies mainly concerned gene therapy of cancer, his latest research interest specifically focused on gene and cell therapy of diabetes due to widespread prevalence of the disease. As being the author of numerous research articles published in the prominent gene therapy journals, Dr. Sanlioglu constantly thrives upon novel genetic discoveries, which might one day make diabetes a treatable disease. One of his ultimate goals is to treat diabetes using 3rd generation of HIV-based Lentiviral vectors with antidiabetic properties.

For this purpose, genes with antidiabetic potential like VIP or GLP-1 have been cloned into the most advanced gene therapy vectors currently available. Dr. Sanlioglu has currenlty been working as a full professor at the Human Gene and Cell Therapy Center of Akdeniz University Hospitals and Clinics.

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Home Page of the Human Gene and Cell Therapy Center

WHERE DO WE GET ADULT STEM CELLS? – Stem cell

There are several ways adult stem cells can be isolated, most of which are being actively explored by our researchers.

1) From the body itself: Scientists are discovering that many tissues and organs contain a small number of adult stem cells that help maintain them. Adult stem cells have been found in the brain, bone marrow, blood vessels, skeletal muscle, skin, teeth, heart, gut, liver, and other (although not all) organs and tissues. They are thought to live in a specific area of each tissue, where they may remain dormant for years, dividing and creating new cells only when they are activated by tissue injury, disease or anything else that makes the body need more cells.

Adult stem cells can be isolated from the body in different ways, depending on the tissue. Blood stem cells, for example, can be taken from a donors bone marrow, from blood in the umbilical cord when a baby is born, or from a persons circulating blood. Mesenchymal stem cells, which can make bone, cartilage, fat, fibrous connective tissue, and cells that support the formation of blood can also be isolated from bone marrow. Neural stem cells (which form the brains three major cell types) have been isolated from the brain and spinal cord. Research teams at Childrens, headed by leading scientists Stuart Orkin, MD and William Pu, MD, both affiliate members of the Stem Cell Program, recently isolated cardiac stem cells from the heart.

Isolating adult stem cells, however, is just the first step. The cells then need to be grown to large enough numbers to be useful for treatment purposes. The laboratory of Leonard Zon, MD, director of the Stem Cell Program, has developed a technique for boosting numbers of blood stem cells thats now in Phase I clinical testing.

2) From amniotic fluid: Amniotic fluid, which bathes the fetus in the womb, contains fetal cells including mesenchymal stem cells, which are able to make a variety of tissues. Many pregnant women elect to have amniotic fluid drawn to test for chromosome defects, the procedure known as amniocentesis. This fluid is normally discarded after testing, but Childrens Hospital Boston surgeon Dario Fauza, MD, a Principal Investigator at Childrens and an affiliate member of the Stem Cell Program, has been investigating the idea of isolating mesenchymal stem cells and using them to grow new tissues for babies who have birth defects detected while they are still in the womb, such as congenital diaphragmatic hernia. These tissues would match the baby genetically, so would not be rejected by the immune system, and could be implanted either in utero or after the baby is born.

3) From pluripotent stem cells: Because embryonic stem cells and induced pluripotent cells (iPS cells), which are functionally similar, are able to create all types of cells and tissues, scientists at Childrens and elsewhere hope to use them to produce many different kinds of adult stem cells. Laboratories around the world are testing different chemical and mechanical factors that might prod embryonic stem cells or iPS cells into forming a particular kind of adult stem cell. Adult stem cells made in this fashion would potentially match the patient genetically, eliminating both the problem of tissue rejection and the need for toxic therapies to suppress the immune system.

4) From other adult stem cells: A number of research groups have reported that certain kinds of adult stem cells can transform, or differentiate, into apparently unrelated cell types (such as brain stem cells that differentiate into blood cells or blood-forming cells that differentiate into cardiac muscle cells). This phenomenon, called transdifferentiation, has been reported in some animals. However, its still far from clear how versatile adult stem cells really are, whether transdifferentiation can occur in human cells, or whether it could be made to happen reliably in the lab.

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WHERE DO WE GET ADULT STEM CELLS? - Stem cell

World Stem Cell Summit

ATLANTA, GEORGIA, USA!

11th World Stem Cell Summit 2nd RegMed Capital Conference Hyatt Regency Atlanta

Collect Opportunities Expand Knowledge Forge Collaborations Deliver Cures

The mandatory, big-picture conference with one-on-one partnering

EXPAND KNOWLEDGE FORGE COLLABORATIONS DELIVER CURES

RegMed Capital Conference

Connecting companies to investors, bringing start-up angel capital and new money into the field. Advancing investment and commercialization, with the overarching purpose of accelerating cures. Investment bankers, analysts, venture groups, angel networks, family funds are attending.

Numerous networking events to make the best connections to advance your goals

7 Tracks- COMPREHENSIVE, TIMELY & DIRECT

ATLANTA, GEORGIA, USA! 11th World Stem Cell Summit 2nd RegMed Capital Conference Hyatt Regency Atlanta

Collect Opportunities Expand Knowledge Forge Collaborations Deliver Cures

Anthony Atala, MD

Wake Forest Institute for Regenerative Medicine

Scott Gottlieb, MD

T.R Winston & Company

Outi Hovatta, MD, PhD

Karolinska Institutet

Paul Knoepfler, PhD

UC Davis School of Medicine

Joanne Kurtzberg, MD

Carolinas Cord Blood Bank at Duke

Jeanne F. Loring, PhD

Center for Regenerative Medicine The Scripps Research Institute

Chris Mason, MBBS, PhD, FRCS

University College London

Michael H. May, PhD

Centre for Commercialization of Regenerative Medicine (CCRM)

Todd C. McDevitt, PhD

Gladstone Institute of Cardiovascular Disease

C. Randal Mills, PhD

California Institute for Regenerative Medicine (CIRM)

Linda Myers

BioBridge Global

Norio Nakatsuji, DSc

Institute for Integrated Cell-Material Science (iCeMS) Kyoto University

Jan A. Nolta, PhD

Stem Cell Program and Institute for Regenerative Cures University of California, Davis

J. Peter Rubin, MD

University of Pittsburgh

Steven L. Stice, PhD

University of Georgia

Susan L. Solomon

New York Stem Cell Foundation

Johnna Temenoff, PhD

Georgia Institute of Technology

Andre Terzic, MD, PhD

Center for Regenerative Medicine Mayo Clinic

Yuzo Toda

FUJIFILM Corporation

Karl Tryggvason, MD, PhD

Karolinska Institutet

Edmund K. Ned Waller, MD, PhD

Emory School of Medicine

Paul Wolpe, PhD

Emory University

Claudia Zylberberg, PhD

Akron Biotechnology, LLC

The 2014 World Stem Cell Summit and REGMED Capital Conference in San Antonio were a bonanza for StemBioSys. As a growing company in the stem cell space, preparing to launch our first product in 2015, the connections and ongoing collaborations that we made at these meetings has already exhibited a multifold return on investment in spreading the word about SBS and uncovering several new opportunities for business and product development.

-Bob Hutchens, President & CEO

Mayo Clinic has embraced regenerative medicine as a strategic investment in the future of healthcare. The World Stem Cell Summit brings together the regenerative medicine community, and sets the stage for global collaboration. Our team at Mayo Clinic is honored to lead it as an organizer again this year.

"The World Stem Cell Summit is a meeting of the stem cell minds, bringing Policy, science, industry, advocates and clinicians together in a unique and powerful forum. The Summit is of great value to researchers by providing a context for stem cell science that is not found at other meetings."

The 2014 World Stem Cell Summit and REGMED Capital Conference in San Antonio were a bonanza for StemBioSys. As a growing company in the stem cell space, preparing to launch our first product in 2015, the connections and ongoing collaborations that we made at these meetings has already exhibited a multifold return on investment in spreading the word about SBS and uncovering several new opportunities for business and product development. We are looking forward to the 2015 meetings in Atlanta.

- Bob Hutchens,President & CEO

Mayo Clinic has embraced regenerative medicine as a strategic investment in the future of healthcare. The World Stem Cell Summit brings together the regenerative medicine community, and sets the stage for global collaboration. Our team at Mayo Clinic is honored to lead it as an organizer again this year.

- Dr. Andre Terzic

The World Stem Cell Summit is a meeting of the stem cell minds, bringing Policy, science, industry, advocates and clinicians together in a unique and powerful forum. The Summit is of great value to researchers by providing a context for stem cell science that is not found at other meetings.

- Sally Temple,Ph.D.

The World Stem Cell Summit and the RegMed Capital Conference offers a number of key opportunities to advance progress for sponsors, exhibitors, and advertisers of all sizes, while we collectively support the advancement of the field.

Request a sponsorship kit today.

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World Stem Cell Summit

Embryonic stem cell – ScienceDaily

Embryonic stem cells (ESCs) are stem cells derived from the undifferentiated inner mass cells of a human embryo.

Embryonic stem cells are pluripotent, meaning they are able to grow (i.e. differentiate) into all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm.

In other words, they can develop into each of the more than 200 cell types of the adult body as long as they are specified to do so.

Embryonic stem cells are distinguished by two distinctive properties: their pluripotency, and their ability to replicate indefinitely.

ES cells are pluripotent, that is, they are able to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm.

These include each of the more than 220 cell types in the adult body.

Pluripotency distinguishes embryonic stem cells from adult stem cells found in adults; while embryonic stem cells can generate all cell types in the body, adult stem cells are multipotent and can produce only a limited number of cell types.

Additionally, under defined conditions, embryonic stem cells are capable of propagating themselves indefinitely.

This allows embryonic stem cells to be employed as useful tools for both research and regenerative medicine, because they can produce limitless numbers of themselves for continued research or clinical use.

Because of their plasticity and potentially unlimited capacity for self-renewal, ES cell therapies have been proposed for regenerative medicine and tissue replacement after injury or disease.

Diseases that could potentially be treated by pluripotent stem cells include a number of blood and immune-system related genetic diseases, cancers, and disorders; juvenile diabetes;

Parkinson's; blindness and spinal cord injuries.

Besides the ethical concerns of stem cell therapy, there is a technical problem of graft-versus-host disease associated with allogeneic stem cell transplantation.

However, these problems associated with histocompatibility may be solved using autologous donor adult stem cells, therapeutic cloning, stem cell banks or more recently by reprogramming of somatic cells with defined factors (e.g. induced pluripotent stem cells).

Other potential uses of embryonic stem cells include investigation of early human development, study of genetic disease and as in vitro systems for toxicology testing.

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Embryonic stem cell - ScienceDaily

1. Embryonic Stem Cells [Stem Cell Information]

by Junying Yu* and James A. Thomson**

Human embryonic stem (ES) cells capture the imagination because they are immortal and have an almost unlimited developmental potential (Fig. 1.1: How hESCs are derived). After many months of growth in culture dishes, these remarkable cells maintain the ability to form cells ranging from muscle to nerve to bloodpotentially any cell type that makes up the body. The proliferative and developmental potential of human ES cells promises an essentially unlimited supply of specific cell types for basic research and for transplantation therapies for diseases ranging from heart disease to Parkinson's disease to leukemia. Here we discuss the origin and properties of human ES cells, their implications for basic research and human medicine, and recent research progress since August 2001, when President George W. Bush allowed federal funding of this research for the first time. A previous report discussed progress prior to June 17, 2001 (/info/scireport/.)

Figure 1.1. How Human Embryonic Stem Cells are Derived.

( 2006 Terese Winslow)

Embryonic stem cells are derived from embryos at a developmental stage before the time that implantation would normally occur in the uterus. Fertilization normally occurs in the oviduct, and during the next few days, a series of cleavage divisions occur as the embryo travels down the oviduct and into the uterus. Each of the cells (blastomeres) of these cleavage-stage embryos are undifferentiated, i.e. they do not look or act like the specialized cells of the adult, and the blastomeres are not yet committed to becoming any particular type of differentiated cell. Indeed, each of these blastomeres has the potential to give rise to any cell of the body. The first differentiation event in humans occurs at approximately five days of development, when an outer layer of cells committed to becoming part of the placenta (the trophectoderm) separates from the inner cell mass (ICM). The ICM cells have the potential to generate any cell type of the body, but after implantation, they are quickly depleted as they differentiate to other cell types with more limited developmental potential. However, if the ICM is removed from its normal embryonic environment and cultured under appropriate conditions, the ICM-derived cells can continue to proliferate and replicate themselves indefinitely and still maintain the developmental potential to form any cell type of the body (quot;pluripotencyquot;; see Fig. 1.2: Characteristics of ESCs). These pluripotent, ICM-derived cells are ES cells.

Figure 1.2.Characteristics of Embryonic Stem Cells.

( 2006 Terese Winslow)

The derivation of mouse ES cells was first reported in 1981,1,2 but it was not until 1998 that derivation of human ES cell lines was first reported.3 Why did it take such a long time to extend the mouse results to humans? Human ES cell lines are derived from embryos produced by in vitro fertilization (IVF), a process in which oocytes and sperm are placed together to allow fertilization to take place in a culture dish. Clinics use this method to treat certain types of infertility, and sometimes, during the course of these treatments, IVF embryos are produced that are no longer needed by the couples for producing children. Currently, there are nearly 400,000 IVF-produced embryos in frozen storage in the United States alone,4 most of which will be used to treat infertility, but some of which (~2.8%) are destined to be discarded. IVF-produced embryos that would otherwise have been discarded were the sources of the human ES cell lines derived prior to President Bush's policy decision of August 2001. These human ES cell lines are now currently eligible for federal funding. Although attempts to derive human ES cells were made as early as the 1980s, culture media for human embryos produced by IVF were suboptimal. Thus, it was difficult to culture single-cell fertilized embryos long enough to obtain healthy blastocysts for the derivation of ES cell lines. Also, species-specific differences between mice and humans meant that experience with mouse ES cells was not completely applicable to the derivation of human ES cells. In the 1990s, ES cell lines from two non-human primates, the rhesus monkey5 and the common marmoset,6 were derived, and these offered closer models for the derivation of human ES cells. Experience with non-human primate ES cell lines and improvements in culture medium for human IVF-produced embryos led rapidly to the derivation of human ES cell lines in 1998.3

Because ES cells can proliferate without limit and can contribute to any cell type, human ES cells offer an unprecedented access to tissues from the human body. They will support basic research on the differentiation and function of human tissues and provide material for testing that may improve the safety and efficacy of human drugs (Figure 1.3: Promise of SC Research).7,8 For example, new drugs are not generally tested on human heart cells because no human heart cell lines exist. Instead, researchers rely on animal models. Because of important species-specific differences between animal and human hearts, however, drugs that are toxic to the human heart have occasionally entered clinical trials, sometimes resulting in death. Human ES cell-derived heart cells may be extremely valuable in identifying such drugs before they are used in clinical trials, thereby accelerating the drug discovery process and leading to safer and more effective treatments.911 Such testing will not be limited to heart cells, but to any type of human cell that is difficult to obtain by other sources.

Figure 1.3: The Promise of Stem Cell Research.

( 2006 Terese Winslow)

Human ES cells also have the potential to provide an unlimited amount of tissue for transplantation therapies to treat a wide range of degenerative diseases. Some important human diseases are caused by the death or dysfunction of one or a few cell types, e.g., insulin-producing cells in diabetes or dopaminergic neurons in Parkinson's disease. The replacement of these cells could offer a lifelong treatment for these disorders. However, there are a number of challenges to develop human ES cell-based transplantation therapies, and many years of basic research will be required before such therapies can be used to treat patients. Indeed, basic research enabled by human ES cells is likely to impact human health in ways unrelated to transplantation medicine. This impact is likely to begin well before the widespread use of ES cells in transplantation and ultimately could have a more profound long-term effect on human medicine. Since August 2001, improvements in culture of human ES cells, coupled with recent insights into the nature of pluripotency, genetic manipulation of human ES cells, and differentiation, have expanded the possibilities for these unique cells.

Mouse ES cells and human ES cells were both originally derived and grown on a layer of mouse fibroblasts (called quot;feeder cellsquot;) in the presence of bovine serum. However, the factors that sustain the growth of these two cell types appear to be distinct. The addition of the cytokine, leukemia inhibitory factor (LIF), to serum-containing medium allows mouse ES cells to proliferate in the absence of feeder cells. LIF modulates mouse ES cells through the activation of STAT3 (signal transducers and activators of transcription) protein. In serum-free culture, however, LIF alone is insufficient to prevent mouse ES cells from differentiating into neural cells. Recently, Ying et al. reported that the combination of bone morphogenetic proteins (BMPs) and LIF is sufficient to support the self-renewal of mouse ES cells.12 The effects of BMPs on mouse ES cells involve induction of inhibitor of differentiation (Id) proteins, and inhibition of extracellular receptor kinase (ERK) and p38 mitogen-activated protein kinases (MAPK).12,13 However, LIF in the presence of serum is not sufficient to promote the self-renewal of human ES cells,3 and the LIF/STAT3 pathway appears to be inactive in undifferentiated human ES cells.14,15 Also, the addition of BMPs to human ES cells in conditions that would otherwise support ES cells leads to the rapid differentiation of human ES cells.16,17

Several groups have attempted to define growth factors that sustain human ES cells and have attempted to identify culture conditions that reduce the exposure of human ES cells to non human animal products. One important growth factor, bFGF, allows the use of a serum replacement to sustain human ES cells in the presence of fibroblasts, and this medium allowed the clonal growth of human ES cells.18 A quot;feeder-freequot; human ES cell culture system has been developed, in which human ES cells are grown on a protein matrix (mouse Matrigel or Laminin) in a bFGF-containing medium that is previously quot;conditionedquot; by co-culture with fibroblasts.19 Although this culture system eliminates direct contact of human ES cells with the fibroblasts, it does not remove the potential for mouse pathogens being introduced into the culture via the fibroblasts. Several different sources of human feeder cells have been found to support the culture of human ES cells, thus removing the possibility of pathogen transfer from mice to humans.2023 However, the possibility of pathogen transfer from human to human in these culture systems still remains. More work is still needed to develop a culture system that eliminates the use of fibroblasts entirely, which would also decrease much of the variability associated with the current culture of human ES cells. Sato et al. reported that activation of the Wnt pathway by 6-bromoindirubin3'-oxime (BIO) promotes the self-renewal of ES cells in the presence of bFGF, Matrigel, and a proprietary serum replacement product.24 Amit et al. reported that bFGF, TGF, and LIF could support some human ES cell lines in the absence of feeders.25 Although there are some questions about how well these new culture conditions will work for different human ES cell lines, there is now reason to believe that defined culture conditions for human ES cells, which reduce the potential for contamination by pathogens, will soon be achieved*.

Once a set of defined culture conditions is established for the derivation and culture of human ES cells, challenges to improve the medium will still remain. For example, the cloning efficiency of human ES cellsthe ability of a single human ES cell to proliferate and become a colonyis very low (typically less than 1%) compared to that of mouse ES cells. Another difficulty is the potential for accumulation of genetic and epigenetic changes over prolonged periods of culture. For example, karyotypic changes have been observed in several human ES cell lines after prolonged culture, and the rate at which these changes dominate a culture may depend on the culture method.26,27 The status of imprinted (epigenetically modified) genes and the stability of imprinting in various culture conditions remain completely unstudied in human ES cells**. The status of imprinted genes can clearly change with culture conditions in other cell types.28,29 These changes present potential problems if human ES cells are to be used in cell replacement therapy, and optimizing medium to reduce the rate at which genetic and epigenetic changes accumulate in culture represents a long-term endeavor. The ideal human ES cell medium, then, (a) would be cost-effective and easy to use so that many more investigators can use human ES cells as a research tool; (b) would be composed entirely of defined components not of animal origin; (c) would allow cell growth at clonal densities; and (d) would minimize the rate at which genetic and epigenetic changes accumulate in culture. Such a medium will be a challenge to develop and will most likely be achieved through a series of incremental improvements over a period of years.

Among all the newly derived human ES cell lines, twelve lines have gained the most attention. In March 2004, a South Korean group reported the first derivation of a human ES cell line (SCNT-hES-1) using the technique of somatic cell nuclear transfer (SCNT). Human somatic nuclei were transferred into human oocytes (nuclear transfer), which previously had been stripped of their own genetic material, and the resultant nuclear transfer products were cultured in vitro to the blastocyst stage for ES cell derivation.30*** Because the ES cells derived through nuclear transfer contain the same genetic material as that of the nuclear donor, the intent of the procedure is that the differentiated derivatives would not be rejected by the donor's immune system if used in transplantation therapy. More recently, the same group reported the derivation of eleven more human SCNT-ES cell lines*** with markedly improved efficiency (16.8 oocytes/line vs. 242 oocytes/line in their previous report).31*** However, given the abnormalities frequently observed in cloned animals, and the costs involved, it is not clear how useful this procedure will be in clinical applications. Also, for some autoimmune diseases, such as type I diabetes, merely providing genetically-matched tissue will be insufficient to prevent immune rejection.

Additionally, new human ES cell lines were established from embryos with genetic disorders, which were detected during the practice of preimplantation genetic diagnosis (PGD). These new cell lines may provide an excellent in vitro model for studies on the effects that the genetic mutations have on cell proliferation and differentiation.32

* Editor's note: Papers published since this writing report defined culture conditions for human embryonic stem cells. See Ludwig et al., Nat. Biotech 24: 185187, 2006; and Lu et al., PNAS 103:56885693, 2006.08.14.

** Editor's note: Papers published since the time this chapter was written address this: see Maitra et al., Nature Genetics 37, 10991103, 2005; and Rugg-Gunn et al., Nature Genetics 37:585587, 2005.

*** Editor's note: Both papers referenced in 30 and 31 were later retracted: see Science 20 Jan 2006; Vol. 311. No. 5759, p. 335.

To date, more than 120 human ES cell lines have been established worldwide,33* 67 of which are included in the National Institutes of Health (NIH) Registry. As of this writing, 21 cell lines are currently available for distribution, all of which have been exposed to animal products during their derivation. Although it has been eight years since the initial derivation of human ES cells, it is an open question as to the extent that independent human ES cell lines differ from one another. At the very least, the limited number of cell lines cannot represent a reasonable sampling of the genetic diversity of different ethnic groups in the United States, and this has consequences for drug testing, as adverse reactions to drugs often reflect a complex genetic component. Once defined culture conditions are well established for human ES cells, there will be an even more compelling need to derive additional cell lines.

* Editor's note: One recent report now estimates 414 hESC lines, see Guhr et al., http://www.StemCells.com early online version for June 15, 2006: quot;Current State of Human Embryonic Stem Cell Research: An Overview of Cell Lines and their Usage in Experimental Work.quot;

The ability of ES cells to develop into all cell types of the body has fascinated scientists for years, yet remarkably little is known about factors that make one cell pluripotent and another more restricted in its developmental potential. The transcription factor Oct4 has been used as a key marker for ES cells and for the pluripotent cells of the intact embryo, and its expression must be maintained at a critical level for ES cells to remain undifferentiated.34 The Oct4 protein itself, however, is insufficient to maintain ES cells in the undifferentiated state. Recently, two groups identified another transcription factor, Nanog, that is essential for the maintenance of the undifferentiated state of mouse ES cells.35,36 The expression of Nanog decreased rapidly as mouse ES cells differentiated, and when its expression level was maintained by a constitutive promoter, mouse ES cells could remain undifferentiated and proliferate in the absence of either LIF or BMP in serum-free medium.12 Nanog is also expressed in human ES cells, though at a much lower level compared to that of Oct4, and its function in human ES cells has yet to be examined.

By comparing gene expression patterns between different ES cell lines and between ES cells and other cell types such as adult stem cells and differentiated cells, genes that are enriched in the ES cells have been identified. Using this approach, Esg-1, an uncharacterized ES cell-specific gene, was found to be exclusively associated with pluripotency in the mouse.37 Sperger et al. identified 895 genes that are expressed at significantly higher levels in human ES cells and embryonic carcinoma cell lines, the malignant counterparts to ES cells.38 Sato et al. identified a set of 918 genes enriched in undifferentiated human ES cells compared with their differentiated counterparts; many of these genes were shared by mouse ES cells.39 Another group, however, found 92 genes, including Oct4 and Nanog, enriched in six different human ES cell lines, which showed limited overlap with those in mouse ES cell lines.40 Care must be taken to interpret these data, and the considerable differences in the results may arise from the cell lines used in the experiments, methods to prepare and maintain the cells, and the specific methods used to profile gene expression.

Since establishing human ES cells in 1998, scientists have developed genetic manipulation techniques to determine the function of particular genes, to direct the differentiation of human ES cells towards specific cell types, or to tag an ES cell derivative with a certain marker gene. Several approaches have been developed to introduce genetic elements randomly into the human ES cell genome, including electroporation, transfection by lipid-based reagents, and lentiviral vectors.4144 However, homologous recombination, a method in which a specific gene inside the ES cells is modified with an artificially introduced DNA molecule, is an even more precise method of genetic engineering that can modify a gene in a defined way at a specific locus. While this technology is routinely used in mouse ES cells, it has recently been successfully developed in human ES cells (See chapter 4: Genetically Modified Stem Cells), thus opening new doors for using ES cells as vehicles for gene therapy and for creating in vitro models of human genetic disorders such as Lesch-Nyhan disease.45,46 Another method to test the function of a gene is to use RNA interference (RNAi) to decrease the expression of a gene of interest (see Figure 1.4: RNA interference). In RNAi, small pieces of double-stranded RNA (siRNA; small interfering RNA) are either chemically synthesized and introduced directly into cells, or expressed from DNA vectors. Once inside the cells, the siRNA can lead to the degradation of the messenger RNA (mRNA), which contains the exact sequence as that of the siRNA. mRNA is the product of DNA transcription and normally can be translated into proteins. RNAi can work efficiently in somatic cells, and there has been some progress in applying this technology to human ES cells.4749

Figure 1.4. How RNAi Can Be Used To Modify Stem Cells.

( 2006 Terese Winslow)

The pluripotency of ES cells suggests possible widespread uses for these cells and their derivatives. The ES cell-derived cells can potentially be used to replace or restore tissues that have been damaged by disease or injury, such as diabetes, heart attacks, Parkinson's disease or spinal cord injury. The recent developments in these particular areas are discussed in detail in other chapters, and Table 1 summarizes recent publications in the differentiation of specific cell lineages.

The differentiation of ES cells also provides model systems to study early events in human development. Because of possible harm to the resulting child, it is not ethically acceptable to experimentally manipulate the postimplantation human embryo. Therefore, most of what is known about the mechanisms of early human embryology and human development, especially in the early postimplantation period, is based on histological sections of a limited number of human embryos and on analogy to the experimental embryology of the mouse. However, human and mouse embryos differ significantly, particularly in the formation, structure, and function of the fetal membranes and placenta, and the formation of an embryonic disc instead of an egg cylinder.5052 For example, the mouse yolk sac is a well-vascularized, robust, extraembryonic organ throughout gestation that provides important nutrient exchange functions. In humans, the yolk sac also serves important early functions, including the initiation of hematopoiesis, but it becomes essentially a vestigial structure at later times or stages in gestation. Similarly, there are dramatic differences between mouse and human placentas, both in structure and function. Thus, mice can serve in a limited capacity as a model system for understanding the developmental events that support the initiation and maintenance of human pregnancy. Human ES cell lines thus provide an important new in vitro model that will improve our understanding of the differentiation of human tissues, and thus provide important insights into processes such as infertility, pregnancy loss, and birth defects.

Human ES cells are already contributing to the study of development. For example, it is now possible to direct human ES cells to differentiate efficiently to trophoblast, the outer layer of the placenta that mediates implantation and connects the conceptus to the uterus.17,53 Another use of human ES cells is for the study of germ cell development. Cells resembling both oocytes and sperm have been successfully derived from mouse ES cells in vitro.5456 Recently, human ES cells have also been observed to differentiate into cells expressing genes characteristic of germ cells.57 Thus it may also be possible to derive oocytes and sperm from human ES cells, allowing the detailed study of human gametogenesis for the first time. Moreover, human ES cell studies are not limited to early differentiation, but are increasingly being used to understand the differentiation and functions of many human tissues, including neural, cardiac, vascular, pancreatic, hepatic, and bone (see Table 1). Moreover, transplantation of ES-derived cells has offered promising results in animal models.5867

Although scientists have gained more insights into the biology of human ES cells since 2001, many key questions remain to be addressed before the full potential of these unique cells can be realized. It is surprising, for example, that mouse and human ES cells appear to be so different with respect to the molecules that mediate their self-renewal, and perhaps even in their developmental potentials. BMPs, for example, in combination with LIF, promote the self-renewal of mouse ES cells. But in conditions that would otherwise support undifferentiated proliferation, BMPs cause rapid differentiation of human ES cells. Also, human ES cells differentiate quite readily to trophoblast, whereas mouse ES cells do so poorly, if at all. One would expect that at some level, the basic molecular mechanisms that control pluripotency would be conserved, and indeed, human and mouse ES cells share the expression of many key genes. Yet we remain remarkably ignorant about the molecular mechanisms that control pluripotency, and the nature of this remarkable cellular state has become one of the central questions of developmental biology. Of course, the other great challenge will be to continue to unravel the factors that control the differentiation of human ES cells to specific lineages, so that ES cells can fulfill their tremendous promise in basic human biology, drug screening, and transplantation medicine.

We thank Lynn Schmidt, Barbara Lewis, Sangyoon Han and Deborah J. Faupel for proofreading this report.

Notes:

* Genetics and Biotechnology Building, Madison, WI 53706, Email: jyu@primate.wisc.edu.

** John D. MacArthur Professor, Department of Anatomy, University of WisconsinMadison Medical School, The Genome Center of Wisconsin, and The Wisconsin National Primate Research Center, Madison, WI 53715, Email: thomson@primate.wisc.edu.

Introduction|Table of Contents|Chapter 2

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1. Embryonic Stem Cells [Stem Cell Information]