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Wednesday, October 6, 2010


Human ESC and iPSC cultures. The hESC
lines used in this study were H1, H7, and H9
[24]; nine Harvard Stem Cell Institute hESC
lines [25] and thirteen hESC lines derived at
Advanced Cell Technology[26,27] (See Table1
and supplementary Table 1). The hiPSC lines
used were iPS(IMR90)-1, iPS(Foreskin)1-1,
iPS(Foreskin)4-1, and 4-3, which were generated
using Thomson’s four factors (Oct-4, Sox-2,
Nanog and Lin28) with Lentiviral vector [1]; rvhiPS01,
rv-hiPS02, rv-hiPS03 and rv-hiPS04,
which were derived by using Yamanaka’s four
factors (Oct-4, Sox-2, c-Myc and Klf4) with
retroviral vector [6]. Cells were cultured on
mitomycin-treated feeder MEFs in hESC
medium supplemented with 20% Knockout
Serum Replacement (Invitrogen), 10 ng/ml
bFGF (Stemgent), ��-mercaptoethanol (Sigma),
NEAA, GlutaMax-I and
penicillin/streptomycin(Invitrogen). Cells were
fed with fresh medium daily and confluent
hESC/hiPSC cultures were split using 1 mg/ml
collagenase IV in DMEM/F12 (Invitrogen).
Embryoid body (EBs) formation. hESC/hiPSC
cultures were incubated with collagenase IV at
37o C for up to 10 minutes, and the large clumps
of cells were collected. The clumps were washed
once with 5 ml hESC medium. To initiate
differentiation, the cell clumps were resuspended
in EB differentiation medium [Stemline II
(Sigma) supplemented with 50 ng/ml BMP4
(R&D System) and VEGF (Invitrogen)], at a
density of approximately 2× 106 cells per well of
a 6-well ultralow attachment plate (Corning).
Generation of hemangioblasts/BCs.
Hemangioblasts/BCs were generated as
previously described [21,22]. Briefly, EBs were
cultured in differentiation medium for 48 hours,
and then half of the medium replaced with
medium containing BMP4 (50 ng/ml), VEGF
(50 ng/ml) and bFGF (20 ng/ml). After 24-hour
incubation, the EBs were collected, washed once
in 2 ml D-PBS (Invitrogen), and incubated with
1 ml of trypsin/EDTA (Invitrogen) at 37o C for
2-3 minutes. The EBs were dissociated by
pipetting up and down 10 times using a 1000 μl
pipette or until the cell suspension became
homogenous with no visible clumps. An equal
volume of MEF medium was added and the cells
were centrifuged at 210g for 4 minutes. The cell
pellet was resuspended in Stemline II medium to
1-2 �� 106 cells/ml, 5 �� 104 cells plated in 1- 2 ml
of blast growth medium (BGM) [21,22], and
then incubated for up to 6 days.
Characterization of hematopoietic colony
forming capability. Day 6 hiPSC-BCs and
hESC-BCs were purified from blast cell cultures.
1 × 103 blast cells were resuspended in 20 ��l of
Stemline II medium, mixed well with 0.5 ml of
either CFC medium (Stem Cell Technologies,
H4436 only) or BGM (H4436 plus cytokines)
using 2 quick vortex pulses. Cells in semi-solid
CFU assay medium were plated into wells of a
24-well Ultra-low attachment plate for CFU
colony growth. Cells were incubated in a CO2
incubator at 37°C for up to 15 days. The
formation of hematopoietic colonies was
monitored microscopically. On day 15, the
number of hematopoietic colonies in each well
as well as their morphology was recorded. For
the CFU-MK assay, BCs were purified and
plated according to the manufacturer’s
instructions using a MK CFU assay kit (Stem
Cell Technologies, Cat 04973).
Differentiation of hiPSC-BCs and hESC-BCs
into erythroid cells. Erythroid differentiation
was carried out as previously described[23].
Briefly, day 5 blast cells from both hiPSCs and
hESCs were purified and plated into 24-well
plates (30,000 cells/ well) with BGM medium
and allow to expand for 5-7 day or until the
culture become confluent. At the end of each
expansion, cells were collected from each well
and counted. To expand further, equal numbers
of cells from both hiPSC-BCs and hESC-BCs
were replated in BGM medium. A total of three
expansion cycles were performed in this study
(each expansion experiment was carried out in
triplicate). To further differentiate toward
erythroid cells, expanded BGM medium with
cells was mixed at 1:1 ratio with Stemline II +
EPO (3U/ml, Cell Sciences) and cultured for an
additional 6-8 days. Additional Stemline II +
EPO medium was added every 2-3 days. The
erythrocytes were then spun onto glass slides and
stained for benzidine/Giemsa (Sigma) and
erythrocyte marker CD235a (DAKO) as
described previously [23].
Differentiation of endothelial cells from hiPSCBCs
and hESC-BCs. Purified BCs from both
hESCs and hiPSCs were plated into wells of
fibronectin-coated 24-well plate (BD
Biosciences) with EGM-2 medium (Lonza) at
the density of 36,000 cells per well (n=4).
Endothelial cells differentiated from hESCs and
hiPSCs were sub-cultured when they reached
confluence, and were continuously cultured for
up to 30 days. The morphology and growth rate
of endothelial cells were recorded. To test the
endothelial clonogenic capability of BCs derived
from hESCs and hiPSCs, individual blast
colonies were handpicked and plated for
endothelial cell colony formation.
Endothelial markers and cell senescence
staining. The BC-derived endothelial cells were
allowed to grow for 10 days in EGM-2 medium.
The endothelial cells derived from hiPSCs were
trypsinized and plated onto Matrigel (BD
Biosciences) for network structure formation as
described previously[21]. Expression of
endothelial markers CD31(DAKO), VE-Cad
(Cell Signaling Technology) and vWF (DAKO)
on the hiPSC-derived endothelial cells was
confirmed by immunofluorescence staining as
described previously[21]. LDL uptake of hiPSCendothelial
cells was evaluated by culturing the
cells in the presence of Alexa-Fluor 594 Ac-LDL
(Invitrogen) at 10 ��g/ml in EGM-2 for 8 hours,
followed by washing with medium 3 times. The
intracellular fluorescent LDL was examined
using fluorescence microscopy. For detection of
endothelial cell senescence, cells were fixed
between 20 to 30 day days after initiation of
endothelial differentiation and then stained for
endogenous ��-galactosidase using a Cell
Senescence Kit (Cell Signaling Technology)
according to the manufacturer’s manual.
There is considerable excitement that human

induced pluripotent stem cells (hiPSCs) can

serve as a potentially safe and embryo-free

source of patient-specific cells for regenerative

medicine. Since 2007, when hiPSC lines were

first generated by Yu et al[1] and Takahashi et

al[2], a variety of methods have been reported

for reprogramming somatic cells to
pluripotency[3-7]. The ability of hiPSCs to
differentiate into derivatives of all three
embryonic germ layers is well established, and
rapid progress is being made towards controlled
in vitro differentiation of hiPSCs into specific
cell types, precursors as well as differentiated
progenies representing various tissues, such as
heart [8], pancreas [9], liver [10], eye including
retinal pigment epithelium cells(RPE) [11-14],
neuronal [15,16], and endothelial and
hematopoietic lineages [17-20]. Although these
studies clearly suggest a similar differentiation
potential between hiPSCs and human embryonic
stem cells (hESC), it is unclear whether they can
be expanded into homogeneous cell populations
suitable for use in drug discovery and clinical
We have developed an efficient method to
reproducibly generate large numbers of
bipotential progenitors—known as
hemangioblasts 􀃭 from multiple hESC lines
using an in vitro differentiation system [21,22].
These blast cells (BCs) expressed gene
signatures characteristic of hemangioblasts, and
could be differentiated into multiple
hematopoietic lineages as well as into
endothelial cells that could repair ischemic
vasculature in vivo[21]. Using hESC-derived
hemangioblasts/BCs as intermediates, we have
also generated functional oxygen-carrying
erythrocytes on a large scale from multiple hESC
lines, demonstrating the robust expansion
capability of these cells[23]. This system
provides an excellent model for evaluation and
comparison of hiPSC derivatives to their hESC
counterparts. In the present study, we
successfully generated BCs, endothelial cells,
and hematopoietic cells from multiple wellcharacterized
hiPSC lines. We further compared
the functional characteristics of BCs derived
from hESCs and hiPSCs, and found that
hemangioblastic derivatives generated from
these hiPSC lines display abnormal molecular
and/or cellular processes compared to their
corresponding hESC counterparts. Similarly,
RPE cells derived from hiPSCs begin senescing
in the first passage, indicating the observed
phenomenon is not limited to hemangioblastic

Sunday, May 30, 2010

stem cell

Current uses of Stem Cell

Current uses of Stem Cell

It have many many uses in world....It has many uses in every health problem...........
Every people looking it for sick's remedy

Brain Damage

Stroke and traumatic brain injury lead to cell death, characterized by a loss of neurons and oligodendrocytes within the brain. Healthy adult brains contain neural stem cells, these divide and act to maintain general stem cell numbers or become progenitor cells.
In healthy adult animals, progenitor cells migrate within the brain and function primarily to maintain neuron populations for olfaction (the sense of smell). Interestingly, in pregnancy and after injury, this system appears to be regulated by growth factors and can increase the rate at which new brain matter is formed.

In the case of brain injury, although the reparative process appears to initiate, substantial recovery is rarely observed in adults, suggesting a lack of robustness.
Recently, results from research conducted in rats subjected to stroke, suggested that administration of drugs to increase the stem cell division rate and direct the survival and differentiation of newly formed cells could be successful. In the study referenced below, biological drugs were administered after stroke to activate two key steps in the reparative process.
Findings from this study seem to support a new strategy for the treatment of stroke using a simple elegant approach, aimed at directing recovery from stroke by potentially protecting and/or regenerating new tissue. The authors found that, within weeks, recovery of brain structure is accompanied by recovery of lost limb function suggesting the potential for development of a new class of stroke therapy or brain injury therapy in humans.


Research injecting neural (adult) stem cells into the brains of dogs has shown to be be very successful in treating cancerous tumors. With traditional techniques brain cancer is almost impossible to treat because it spreads so rapidly. Researchers at the Harvard Medical School induced intracranial tumours in rodents.
Then, they injected human neural stem cells. Within days the cells had migrated into the cancerous area and produced cytosine deaminase, an enzyme that converts a non-toxic pro-drug into a chemotheraputic agent. As a result, the injected substance was able to reduce tumor mass by 81 percent. The stem cells neither differentiated nor turned tumorigenic.
Some researchers believe that the key to finding a cure for cancer is to inhibit cancer stem cells, where the cancer tumor originates. Currently, cancer treatments are designed to kill all cancer cells, but through this method, researchers would be able to develop drugs to specifically target these stem cells.

Uses of Stem Cell

Uses of

Stem Cell

The cells multiply tirelessly in laboratory dishes, offering a self-replenishing supply from which scientists hope to grow replacement tissues for people with various diseases, including bone marrow for cancer patients, neurons for people with Alzheimer's disease, and pancreatic cells for people with diabetes

Already, researchers have used the stem cells to grow human heart muscle cells that beat in unison in a laboratory dish, as well as blood cells, blood vessel cells, bone, cartilage, neurons and skeletal muscle.

what is stem cell therapy?

what is stem cell therapy?

Advanced Parkinson's Disease

In the course of progressive or traumatic neurological disorders, there is an irreplaceable loss of cells leading to a gradual loss of organ function. Despite limited and often ineffective attempts to self-repair, the body's neural plasticity is not sufficient to restore its lost circuitry. Classical medical therapy, which attempts to modify the biochemistry of these events, does not prevent or stop the basic progressive cell loss, but in fact may cause additional problems. Cell therapy is the most fascinating revolution in medicine currently underway. Cell therapy results from the understanding of complex molecular biology events triggering cellular division and development.
A century-old dogma attributed to the Spanish neuroscientist Cajal, stated that adult brain neurons can not multiply and repair itself. This dogma was recently refuted with the extraordinary discovery of the presence, in the human brain, of cells able to divide and differentiate. These cells, called neural stem cells, are the progenitors of most central nervous system cells. Most importantly, these cells are the basic seeds in the new field of neural cell therapy.
Parkinson's disease is a neurodegenerative disorder characterized by a profound loss of neurons in the substantia nigra, a small region in the brainstem. This cell loss results in the degeneration of the nigrostriatal dopamine system that regulates motor function. This, in turn, leads to motor dysfunction, consisting of poverty and slowness of voluntary movements, tremor, and stooped posture, rigidity, and gait disturbance. Modern knowledge on the causes of Parkinson's disease indicates that successful functional restoration can be achieved by replacing the deficient dopamine molecule in the damaged area of the brain.
Previous studies using fetal dopamine producing tissue implanted in patients with Parkinson's disease showed partial recovery, with limited reproducibility and efficacy. The major limitations of the fetal transplantation procedure were practical, ethical and there were several safety concerns related to the use of fetal tissue.
The isolation of neural progenitors (or neural-stem) cells has opened the potential for use in studies of brain repair and neurodegenerative disorders. These progenitor cells have an extended self-renewal capacity and possess the potential to give rise to all three major brain cell types. They can grow into a large number of progenitor cells in vitro and can be used as a source of newly formed cells for transplantation. Since they are cultured under specific conditions, critical events such as maturation and differentiation are precisely controlled by growth conditions. Methods have been developed to induce progenitor cells to become dopamine neurons, or several other types of neurons. These induced mature neurons can serve as an excellent source for cell replacement therapy in different clinical conditions, including Parkinson's disease.
Cell therapy for Parkinson's disease using progenitor cells differentiated into mature neurons such as dopamine neurons is currently under intense investigation. Because of the rapidly evolving nature of stem cell science and strict implementation of regulatory guidelines in the use of biological therapies, NeuroGeneration has continued to maintain its pioneering position in pursuing its clinical studies.



Vet-Stem Regenerative Cell Therapy is based on a clinical technology originally licensed from Artecel Inc., Sunnyvale, CA. Original patents from University of Pittsburgh and Duke University.

  • Rationale based on consistent therapeutic success in numerous animal models of disease (see sidebar)
  • Adipose-derived adult stem cells (Vet-Stem Regenerative Cells: VSRC™)
  • Autologous cell therapy
  • Currently used in horses with bowed tendons, ligament injuries, and fractures, and in dogs with osteoarthritis
  • More than 2,000 horses treated since 2003
  • No systemic adverse events reported and <>3-6
  • Demonstrated efficacy with VSRC therapy in horses and dogs
    • Cornell University double-blind, placebo controlled study5
    • Retrospective studies3,4
    • Case studies6
Why use adipose-derived regenerative cells rather than regenerative cells derived from bone marrow?
Adipose-derived regenerative cells are:
  • Readily available source
  • Can be collected in far greater concentrations than those from bone marrow24
  • Able to differentiate into multiple lineages implicating their potential in bone, cartilage, and cardiac repair23 (See figure above)
  • Fractions isolated from adipose tissue contain a heterogeneous mixture of regenerative cells, including:23
    • Mesenchymal stem cells
    • Endothelial progenitor cells
    • Pericytes
    • Immune cells
    • Fibroblasts
    • Other growth factor-secreting bioactive cells
Differences in Regenerative Medicine compared to traditional medicine;
  • Does not rely on a single target receptor or a single pathway for its action
  • Regenerative cell mixture is delivered either directly to the traumatic wound (e.g.: tendonitis, desmitis, fracture) or are delivered systemically (e.g.: liver disease, renal disease)
  • Regenerative cells can differentiate into many tissue types, induce repair, and stimulate regeneration22
  • Regenerative cells "communicate" with the cells of their local environment through paracrine and autocrine modalities, creating the optimal environment for natural healing25
  • Regenerative cells produce a variety of both secreted and cell surface substances that regulate tissue growth, integrity, and function25

What is Stem Cell?

In 1999, Science journal recognized the process of extraction of stem cells from human body, - the third significant discovery in biology of 20 century after opening of DNA and decoding of human’s genome.


There are different methods with which people are trying to treat diseases. Among them surgery and chemical (pills) treatment are the most popular, spread and acknowledged. Comparing to this, Cell treatment is one of the youngest, but dynamically developing approach to treat different diseases and we see a bright future and many interesting discoveries in this field.

The most controversial and challenging in the cell method are experiments with the so called, Stem Cells. Thanks to the Stem Cells you could not only treat such disease as diabetes, oncology, immune deficit, parkinson’s, but also can revive your body for esthetic cosmetology purposes.

So, what is stem cell? Firstly, it is immature cell able to self-rejuvenate and evolve into particular cell of the body. In other words, these stem cells have ability to turn into muscle, bone, nerve and in all of other forms of tissues. This theory is grounded on the idea that billions of peoples’ or animals’ cells grew up merely from one cell which is called zygote (joined male and female gamete cells). This single stem cell consists of information not only about body, but also about scheme of successive development. During the process of growing this impregnate egg-cell divides and gives beginning to the all other different kinds of body’s cells. The genome of this stem cell is situated in so called “zero point”. Mechanisms, which determine specialization of the future cell are still not switched on, which means that potentially the cell can become any kind of cells, depending on body needs.

In the body of adult the stem cells can be found generally in the bone marrow, and in very small amounts in all organs and tissues. The stem cells provide restoration of injured tissues and organs. When they receive signal about defect, they rush through blood vessels to fix a problem. As we noticed above, in this way stem cells can repair practically any damage by changing into certain kind of cells, requested by body. But the problem is that in the adult body the number of these cells is very limited and this number is not enough for the struggle with many serious diseases.

The stem cells medication approach is directly connected to the kind of stem cell being used. This has influence not only on the quality and terms of treatment, but also on the cost. There are different ways in which contemporary medicine can get stem calls. As a rule, they are:
  • embryonic cells - from donor or clone
  • patient own stem cells - from spinal cord
  • hemopoietic stem cells - from umbilical cord blood.

All of the above mentioned methods have their advantages and disadvantages. However, for the best results, most advanced clinics are using different types of embryonic stem cells: hemopoietic, nervous, thymic, muscular, cutaneous, etc. Each specific stem cell type has its own medical effects. The essential results have been achieved through combination of different cell types, depending on mechanisms of certain diseases and stages of their progress, etc.

Nowadays, Switzerland, Russia, Germany, Mexico, Barbados and some other countries are having clinics which propose stem cell transplantation treatment.
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