IF Your not buying PSPM pinch yourself. Continue to Buy ACTC
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ok so it was Tony Golden that told you.
This month is key to the future.
For everyones sake i hope it works out for them.
longs and shorts and everyone in between.
Life is short, credibility is something that is earned in life and not bought and or stolen as this case is with this scam.
Your ice pics are amazing. would be cool for it to go 10 cents a share for you so you could ice climb year round aroun d the globe.
take it easy
juk
How can Anyone believe a Company that makes up its Trademark of repeatedly lying to its Shareholders at every turn. It can not EVEN get a website up, but feels entranced to mention it is Coming "this week' next week' with out furthe delay in a fortnight.
GUARANTEE TO BEING A REPORTING COMPANY
DUAL STOCK EXCHANGE LISTING IN JAN 2009
PAYDATE OF MOLYGOLD TO BE ANNOUNCED
Less than Zero credibility. The Company and this guy is all just a BIG FAT LIE. Walton Castle? C'mon, give me break. Too bad it is not in the states I could get a title search back on it in 48hrs to prove he is not on title. Ask yourself, do billionaires buy 11.99 USED camera batterys off of ebay>? ofcourse not. If the Land was Viable Why would they EVER Sell it>? These Mining Scamms have been going on for over a hundred years in the SW and this is just another example.
Sword,
They {Lumb] does not care whatsoever what You or anyone thinks. He only wants his money from the sheeple that You {owe} him. I/R Joe could easily be Lumb/Santini with a $179.00 software program running thru his pc and into his Magicjack when you call to change his voice. He may have been hung over etc and not his "usual' jovial self.
No need for professionalism when the Game is ALL about to change again anyways.
MASSIVE reverse split is coming and many Long shareholders about to become Long Shareholders in Another Lumb penny boss scam.
juk
Rocks,
thank you for your time. It is good to see that Advanced is working hard on protecting future revenue.
This one looks interesting?
10374512 NONE US20040014206 ADVANCED CELL TECHNOLOGY, INC.
Gynogenetic or androgenetic production of pluripotent cells and cell lines, and use thereof to produce differentiated cells and tissues
01-05-2009 Mail Final Rejection (PTOL - 326)
The one below should come in handy when the Seth Lord asks Jango Phett to raise a Clone Army..
12221060 NONE US20090126032 ADVANCED CELL TECHNOLOGY, INC.
Method to produce cloned embryos and adults from cultured cells
05-23-2009 Case Docketed to Examiner in GAU
roiresearch wrote{ maybe share oasis' retirement from HGLC will help bring our mojo back baby!!}
hhmm, mojo? the Company can get its mojo back by Acting upon Statements made over the past year plus and also performing a massive reverse split asap./
Look for a MASSIVE reverse split,1-10,000 the the reorg you have been waiting for in a new Company with your shares restrictd for two years in that Company. just wait you will get what you have been waiting for.
In any event. the new re-organized Company will still be saddled with oweing $76,000 and counting to COHEN, NORRIS, SCHERER, WEINBERGER & WOLMER
juk
Document Number J09000711597
Status ACTIVE
Case Number 2007 CA 8033 AG
Name of Court 15TH JUD. CIR. PALM BEACH CTY.
File Date 02/25/2009
Date of Entry 04/04/2008
Expiration Date 02/25/2014
Amount Due $76,239.62
Interest Rate 11.00 %
Name And Address of Judgment Creditor (Plaintiff)
COHEN, NORRIS, SCHERER, WEINBERGER & WOLMER
712 U.S. HWY. 1, STE. 400
N. PALM BEACH, FL 33408
Name And Address of Judgment Debtor(s) (Defendant(s))
ASAP GLOBAL, INC.
99 SE MIZNER BLVD., STE. 712
BOCA RATON, FL 33432
Document Number: P93000006265
HUNT GOLD CORPORATION
1903 60TH PLACE E., STE. M9042
BRADENTON, FL 34203
Document Number: P93000006265
Events
There are no events for this filing.
http://sunbiz.org/scripts/jlidet.exe?action=DETLIST&inquiry_number=J09000711597&inquiry_date=047427138896055675&return_number=J09000711597&return_date=047427138896055675
Locks,
This is all a mute point now from what we all learned on May 28th 2009. Very much looking forward to getting rid of the embryo part of Stem Cell research and moving into a new age of specific human skin cells which in turn can be "Voila" into Stem Cells.
The embryo part of Stem Cells has always made Stem Cell research 'dirty and wrong' to 50% or more of the population. The influx of investors who will come to invest in stem cells will triple once this Skin 2 Stem is mainstreamed and adopted? I wonder if the patents for this "new' way of transforming regular skin cells into the building blocks of life was not filed back in march etc? anyone know if 'filings' are public knowledge or is accepted patents only public knowledge?
juk
This is outstanding news. Welwind is going to Remake the Image and develop a brand.
http://www.csirgroup.com/ if you look at the website it is for new Companys etc.
The CSIR Group is your link to the investment community, providing you with a practiced, professional approach and proven experience to maximize your investor relations efforts. Partner with CSIR to maximize your IR impact through actionable insight, strategic introductions and effective communications. In today's markets, working with the right group can make all the difference, as the key to maximizing valuation is relationship-building with the investment community.
I sent Bragg a thank you email for his time and professionalism and did not receive a response back from him as well. i think the people at Welwind only email an exclusive few of Us? i have tried several times over the past to email with Welwind etc and noone has ever written me back? and i have emailed from a work account etc so a pc safety filter should let me thru. but well yeah.
Was the Company paying Bragg 4k a month? or was it 5k a month? Being that the Company has less than 20k left in the bank they could not afford to keep him? Bragg never seemed like a typical i/r flimflam guy. always liked him etc.
So what is next? is it the darkest before the light or red sky in the morning sailor take warning?
juk
The world is changing very fast. Big will not beat small anymore. It will be the fast beating the slow.
Rupert Murdoch - Change - Innovation
The NIH Grant money and CHA U, KSC and Dongyang Corp paid the bills. Well NIH hopefully will be excited about a Grant {paying off}?.
This work was supported by National Institutes of
Health (NIH) grants MH48866 and DC 006501 and
by International Grants from the CHA University,
Korean Stem Cell Research Center, and Dongyang
Corporation Co. in Korea. The authors thank Dr. V.
Morgan (Harvard Partners Center for Genetics and
Genomics), Dr. J. Kim (Harvard Medical School),
andDr. J. Lee (ShippensburgUniversity) formicroarray
analysis and Ms. J. Johnson (Cell Line Genetics)
for karyotyping analysis. R.L. is an employee and
shareholder of Advanced Cell Technology and
a scientific advisor for Stem Cell and Regenerative
Medicine International. K.Y.C. is a shareholder of
CHA Bio and Diostech Co., Ltd., and Stem Cell
and Regenerative Medicine International.
In conclusion,
the system described here eliminates the
potential risks associated with chromosomal
chromosomal
integrations and/or mutations and
may allow the translation of hiPSC technology
into the clinic.
Sword,
would You not be on the defense constantly if you got calls all day calling you a F$%^&* or a C%T^YU etc etc? lol.
You were civil/professional to this guy and he probably did not know what to do. This is most likely a waiter from Lumbs favorite restaurant in south florida or maybe a kid from the dry cleaner living at his house and working out of the home office. They guy is literally in Hell right now.
Ok stop back soon. Maybe an update next week[s]
juk
rocks, I will 2nd your post.
I by no means have a complex Company. but needed an Audited financial statement for 2008 to obatin a license, it cost me 18k and took 6 weeks, all my controller did was work on this stuff with the CPA. I think you or another poster brought up the theory that getting reporting again was/may be a requirement of the five million dollar line of credit?
my juk gut is telling me we see the financials Friday/12 and or following Monday and that Advanced will once again be listed on the BB on or before the July 4th weekend. ok my Crystall ball is geting fuzzy,. gotta wrap it up for the night.
:}
juk
Ssok,
Well said. I am still trying to understand how it is legal for the promoters, family members, friends to LOAD up KNOWING of what is to come in the form of a GIANT Pump, OR for a Company to pay for a Pump only to KNOWINGLY dillute the stock after the pump is over/under way with a forward split. If the Company is Paying for a Promotion do they not have an obligation to approve said Promotion? I mean Red Flag to me is reading this is the GREATEST PICK ever!!!lol.
I have noted the uhhum person's here as well.
juk
NOT IN MY BACKYARD!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!.
Liberals and Conservatives alike love to feel good when it is not in his/her back yard. Put Mouth where Your Money is. Teddy boy is a piece of filth, her murderd Mary Jo and gotaway with it, you want to see where Congress is going to be in a couple years of corruption, nepotism and our theft? Just look at the Commonwealth of Mass all One party and one party rule. 20 Billion dollars for the Big dig in boston did NOTHING for the Commute. The people that it helped the most on the south shore pay ZERO tolls!!!LOL.
dont get me started! lol.
Yeah you certainly did call that last stock...
juk
Ssok.
Bid wacking alright, I saw 10 - 100 share whacks at it in as few minutes and the pps fell all day
./ Always a sure sign of a Pump and Dump when the volume is astronomical right before Promo's come out. Hhhm,. I know,. why not buy like everyone else when it is .005 a share and have the PPS sky rocket? thus draw a huge crowd, instead of DUMPING whilst everyone is trying to buy?
Nah always gotta get the inside play..Another POS company. Maybe we will have a f/s split next week as well?
juk
Rocks,
concise and to the point. just concise,
I do not think you have brain tissue anymore, cyborg i think. silica, wheels/cogs, 850gb mother board and 20gigs of ram after coming back with that info so darn fast!!!lolthanks man.
good day
juk
just a shame. close to the equator, 300 sunny days a year? just a shame.
juk
Rocks,
do they have to file some kind of Reg ABCDEF etc etc to let the SEC know of which exchange? does the share type classification change? meaning, if the Company lets say wanted to go to the BB would it have to file a filing which designated it's intentions to file and meet the the requirements by providing proof of such requirements? or is a Filing stating We/Company intend to move to the BB and a follow up filing with supoporting doc;s thank you for your time./
juk
gymmer,
Who did you Speak with Exactly at LF {individual]? What is his name?
by your posting and information learned from light fare, do you think that the Company has Lied to its shareholders? Where else do you think the Company has secured the funds to proceed with manufacturing, hiring Troutman/Sanders, Hauser Group day to day expenses? as they go from a start up to a player?
02/19/2009 @ 10:19AM
PureSpectrum, Inc. (PINKSHEETS: PSPM) has received initial funding from Emirates International Capital Advisory (EICA) in Dubai pursuant to an equity investment commitment agreement reached in December.
"This transaction with EICA will provide multiple benefits for PureSpectrum and our shareholders," said PureSpectrum president and CEO Lee Vanatta, who met with EICA representatives in Dubai earlier this week during the Middle East Electricity Exhibition and Conferences. "In addition to receiving this initial investment, we have begun working with EICA to identify and capitalize on business opportunities within member nations of the Gulf Coast Council as well as other parts of the Middle East and North Africa.
http://ih.advfn.com/p.php?pid=nmona&cb=1243963841&article=36335964&symbol=NO%5EPSPM
rocks,
thanks for the clarification. I hold regular "buy' schedule.
The selling pressure on this security is truly something of an unfortunate nature. Must be a broker with 100 million position standing on the neck of this security day in and day out to perfectly manage the pps with very little swings, just the right amount of selling pressure IMO. 20 more Business days left in the month! I would hope after he/CEO was wrong about the end of April comment he will be covering his bases for a timely revealing of the full financials. Can I bother you for a link for what is required to move to BB and or Amex both if you know?
thx your time!
juk
are you serious? dman it. wtf is wrong with these people. You place these damn solar panels next to the wells. just like cell phone towers do a 25yr lease etc.
I tell yah. all these politicians talk out the arses' and or both sides of his/her owns mouths. we want green energy but well not in our back yard,. the turbines can not make too much noise, and well i do not like they way they look etc etc et cet ce. bunk!
Rocks,
ok man. hope all is well. when you have a minute. I was thinking of moving my monthly 'buy' date up two weeks. to this Thursday instead.
juk
Rocks,
is the http://www.cell.com/cell-stem-cell/home going to publish a detailed article on the possible breakthru in skin 2 stem cell process developed by Advanced, CHA and Harvard this Friday? I thought I read something like that? or will this article just be updated? http://www.cell.com/cell-stem-cell/fulltext/S1934-5909(09)00214-8
I could have sworn I read that this Friday was going to have a Cell.com article.
if you know thanks,
juk
Gosh I tell yah what, The Chinese are SMART to invest that much in Wind. When a Government can manage influxes in price of so called commoditys it is able to control the ebb and flow of good and bad times.
I have been of the opinion that If Our Country had 10-tw's it would be a domonio effect of fixing so many problems, couple that with Nuke Plants? Never burn an ounce of Coal again. either that and up it another 15-tw's and plaster Arizona, NW and Texas with Solar Panels. The government wants Nationalized Healthcare? hhm, how about Nationalized Utility's? I pay 7 HUNDRED dollars a month for my stupid house in heat/electricty etc 8 Grand a year? I gotta make 12k a year gross too afford it. If I had half that back, i would maybe buy a car, or have my yard landscaped etc etc etc etc etce.
What if every american bassically had an extra 150 bucks a month to spend? We could literally solve EVERY financial problem We have by havng an unlimited energy supply. But the Lobbyists own Our so-called Elected Represenatives. I am not a tree hugger, but being clean with energy and cheap is a win win for everyone.
juk
Good article. All press, mainstream or otherwise isa great tool.
One in Eight U.S. women is diagnosed with breast cancer.
Cup formed, trade sideways til end of September for the handle. in time for the Holidays, a few infomercials and voila--28 cent PPS.
here yah go. To get the Catholic's behind Lanza's/Harvard/Kim's way of Skin to Stems would be ideal.
PONTIFICAL ACADEMY OF SCIENCES
Casina Pio IV
V-00120 VATICAN CITY
TEL. +39 0669883195 - FAX +39 0669885218
E-MAIL: academy.sciences@acdscience.va
http://www.vatican.va/roman_curia/pontifical_academies/acdscien/own/index_10121999.htm
http://www.vatican.va/faq/index_en.htm
http://www.cell.com/cell-stem-cell/fulltext/S1934-5909(09)00214-8
Copyright 2009 Elsevier Inc.. All rights reserved.
Cell Stem Cell, 28 May 2009
doi:10.1016/j.stem.2009.05.005
Brief Report
Add to 2CollabDeliciousDiggRedditFacebookStumbleUponRequest permissionGeneration of Human Induced Pluripotent Stem Cells by Direct Delivery of Reprogramming Proteins
Dohoon Kim1,5,Chun-Hyung Kim1,5,Jung-Il Moon1,Young-Gie Chung3,Mi-Yoon Chang1,Baek-Soo Han1,Sanghyeok Ko1,Eungi Yang1,Kwang Yul Cha4,Robert Lanza3,,andKwang-Soo Kim1,2,4,,
1 Molecular Neurobiology Laboratory, Department of Psychiatry and McLean Hospital, Harvard Medical School, 115 Mill Street, Belmont, MA 02478, USA
2 Harvard Stem Cell Institute, 115 Mill Street, Belmont, MA 02478, USA
3 Stem Cell and Regenerative Medicine International, 381 Plantation Street, Worcester, MA 01605, USA
4 CHA Stem Cell Institute, CHA University, 606-16 Yoeksam 1-dong, Gangnam-gu, Korea
Corresponding author
Corresponding author
5 These authors contributed equally to this work
Main Text
To date, all methods to generate induced pluripotent stem cells (iPSCs) require the use of genetic materials and/or potentially mutagenic molecules. Here we report the generation of stable iPSCs from human fibroblasts by directly delivering four reprogramming proteins (Oct4, Sox2, Klf4, and c-Myc) fused with a cell-penetrating peptide (CPP). These protein-induced human iPSCs (p-hiPSCs) exhibited similarity to human embryonic stem cells (hESCs) in morphology, proliferation, and expression of characteristic pluripotency markers. p-hiPSC lines produced with these recombinant proteins were successfully maintained for more than 35 passages and differentiated into derivatives of all three embryonic germ layers both invitro and in teratomas. This system eliminates the potential risks associated with the use of viruses, DNA transfection, and potentially harmful chemicals and in the future could potentially provide a safe source of patient-specific cells for regenerative medicine.
Over a decade ago, Wilmut and colleagues showed that adult somatic cells could be reprogrammed back to an undifferentiated embryonic state using somatic cell nuclear transfer (SCNT) (Wilmut etal., 1997). However, since that time, attempts to generate patient-specific cells using SCNT have proven unsuccessful (Chung etal., 2009,French etal., 2008). In 2006, a new and less controversial method of reprogramming somatic cells to pluripotency was reported by viral expression of the transcription factors Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006). This and subsequent studies confirmed that mouse and human somatic cells can be reprogrammed to the pluripotent state via viral transduction with the same or similar sets of reprogramming factors (Maherali etal., 2007,Okita etal., 2007,Park etal., 2008,Takahashi etal., 2007,Wernig etal., 2007,Yu etal., 2007). Although the therapeutic potential of iPSCs has been demonstrated in animal models of sickle cell anemia and Parkinson's disease (Hanna etal., 2007,Wernig etal., 2008), these cells contain multiple viral vector integrations that make them unsuitable for human clinical trials. The use of genome-integrating viruses could cause insertional mutagenesis and unpredictable genetic dysfunction (Okita etal., 2007,Yamanaka, 2007).
To address whether it is possible to generate hiPSCs without the use of viral or DNA vectors, we attempted to deliver four reprogramming proteinsOct4, Sox2, Klf4, and c-Mycdirectly into somatic cells. A major hurdle for intracellular delivery of macromolecules such as proteins is their limited ability to cross the cellular membrane (Belting etal., 2005). In 1988, Frankel and Pabo found that the human immunodeficiency virus transactivator of transcription (HIV-TAT) protein can overcome this hurdle with a short basic segment residing at amino acids 4860 that allows this protein to penetrate the cell membrane and activate HIV-specific genes (Frankel etal., 1988,Frankel and Pabo, 1988). This and other naturally occurring peptides capable of overcoming the cell membrane barrier contain a high proportion of basic amino acids (e.g., arginine or lysine) and are known as CPPs (El-Sayed etal., 2009,Ziegler etal., 2005). In order to test our hypothesis that CPP-anchored reprogramming proteins may directly reprogram human somatic cells without genetic manipulation and/or chemical treatments, we first examined whether red fluorescent protein (RFP) fused with a 9 arginine (RFP-9R) (Wender etal., 2000) could penetrate into COS-7 cells and human newborn fibroblasts (HNFs). RFP-9R was efficiently delivered into both cell types within a few hours, even when in the context of whole-cell extracts (see FigureS1 available online). We then generated stable HEK293 cell lines that could express each of thefour human reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) fused with 9R and the myc tag. High expression of these proteins was confirmed in HEK293 cell lines by western blotting analyses (FigureS2 ). When HNFs were treated with cell extracts from the HEK293 cell lines, efficient intracellular translocation of each recombinant protein was observed within 8 hr (Figure1A). Notably, in contrast to RFP-9R, which was translocated to the cytoplasm, it appeared thatmost recombinant reprogramming proteins were translocated to the nucleus, while some remained in the cytoplasm (Figure1A and FigureS1 ).
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Figure1
Generation of Protein-Induced hiPSC Lines by Direct Delivery of Reprogramming Proteins Fused with 9R as a CPP
(A) HNFs were incubated with HEK293 extracts expressing each reprogramming protein and subjected to immunocytochemistry using myc antibodies. Nuclei were counterstained with DAPI.
(B) The schematic protocol depicts a repeated process and the timeline for generating p-hiPSCs from HNFs.
(C) (Top panel) Shown are starting HNFs (first image), morphology after three cycle protein treatments (second image), and increased colony number after six cycles (third image). Approximately half of these iPS-like colonies stained positive for AP; early morphology after p-hiPSC colonies were transferred to MEF is shown (fourth image); and morphology of established p-hiPSC line is shown at passage number 10 (p-hiPS01 [fifth image] and p-hiPS02 [sixth image]). Immunostaining of p-hiPS01 (middle panel) and p-hiPS02 (bottom panel) clones show expression of hESC markers, including AP, SSEA-3, SSEA-4, Oct-4, Nanog, and TRA-1-60. Nuclei were stained with DAPI (blue in second and third row of panel).
(D) Shown is efficiency of reprogrammed colony formation with iPS-like morphology and AP-positive staining after different numbers of the protein treatment cycle. This is the summary of three independent experiments with the standard error.
In an initial series of experiments, 5 105 HNFs were treated with combined total extracts of four HEK293 cell lines for 16 hr (see Protocol 1 in FigureS3 ). After washing, cells were incubated for 6 days in ES media 1 and then transferred onto mouse embryonic feeders (MEFs). The transferred cells were incubated with ES media 2 for up to 4 weeks. Despite numerous attempts, we did not observe the formation of reprogrammed colonies using this protocol. We next treated with the same total extracts for 16 hr followed by washing and incubation with ES media 1 for 8 hr/day for 6 days (Protocol 2 in FigureS3 ). By day 7, most cells did not survive, and no colonies formed after further incubation on MEF. One potential reason for the lack of success is that, in contrast to virus- or other DNA-based methods, the reprogramming factors were not provided continuously and thus were in short supply. Therefore, we tested whether repeated protein treatment cycles (16 hr protein treatment followed by 6 day incubation in ES media 1) (Figure1B) could yield hiPSCs. Using this approach, after three or four rounds of treatment, several colonies with iPSC-like morphology were observed (Figure1C), although none of these colonies showed alkaline phosphatase (AP) activity, suggesting only rudimentary reprogramming. When this procedure was repeated for further cycles, the number of iPSC-like colonies significantly increased, and approximately half of the resulting colonies were AP positive starting from the sixth cycle (Figure1D). In contrast, no such colonies were formed at any stage when extracts of naive HEK293 cells were used. AP-positive colonies with iPSC-like morphology were handpicked and transferred onto MEFs in the presence of ES media 2 and ES media 3 for 7 days each. Five hiPSC-like colonies were established, and two of them were maintained and characterized in this study. These two cell lines (p-hiPS01 and p-hiPS02) have been successfully maintained for more than 35 passages and exhibit morphology similar to that of hESCs, characterized by large nuclei and scant cytoplasm (Figure1C). Overall, the establishment of these hiPSC-like colonies took about 8 weeks, approximately double that seen with viral transduction (Park etal., 2008,Takahashi etal., 2007,Yu etal., 2007). At present, the efficiency of hiPSC generation is significantly lower using this protein-based protocol (about 0.001% of input cells; Figure1D), compared to virus-based protocols (about 0.01% of input cells) (Park etal., 2008,Takahashi etal., 2007,Yu etal., 2007).
In order to determine whether the p-hiPSCs have hESC-like properties, we examined them for expression of markers of pluripotency. As shown in Figure1C, both cell lines prominently expressed ESC markers, including AP, Oct4, Nanog, tumor-rejection antigen (TRA)1-60, stage-specific embryonic antigen (SSEA)-3, and SSEA-4. Quantitative reverse transcription PCR (qRT-PCR) analysis confirmed that both lines expressed endogenous mRNAs of ESC markers: Oct4, Nanog, Sox2, reduced expression 1 (Rex1), growth and differentiation factor 3 (Gdf3), and telomerase reverse transcriptase (hTERT) levels were dramatically higher than those of HNF cells (up to 100-fold, and comparable to hESCs) (Figure2A). The expression patterns of ESC pluripotency markers were indistinguishable from hESCs (H9), strongly suggesting that appropriate epigenetic reprogramming had occurred in the p-hiPSCs. Bisulfite sequencing analyses further showed that the promoter regions of the pluripotency genes Nanog and Oct4 were significantly demethylated in both p-hiPSC lines and the hESC H9 line, whereas the same regions were densely methylated in the parental HNF cells (Figure2C). hiPSC lines from the starting HNFs were also generated using retroviral vectors expressing the same four reprogramming factors (Park etal., 2008,Takahashi etal., 2007,Yu etal., 2007). These cells displayed similar characteristics and properties as the p-hiPSCs (FigureS4 ), and one of them (rv-hiPS01) was used as a control. When global gene expression was compared using the Affymetrix Array U133 Plus 2.0, analyzing over 47,000 human transcripts, both p-hiPSC and rv-hiPS01 showed high similarity to hES H9, but not to HNFs (Figure2B, FigureS5 ). Since hES H9 and rv-hiPS01 were used as control cell lines, it was important to rule out the possibility that the new p-hiPSCs were derived from contaminating cells. RT-PCR analyses detected all four transgene mRNAs in rv-hiPS01 cells, but not in p-hiPS01 and p-hiPS02 cell lines (FigureS6 ). Furthermore, DNA fingerprinting demonstrated that the patterns of both p-hiPSC lines and rv-hiPS01 cells were identical to the parental HNF cells but different from those of the hES (H9) cells and HEK293 cells (FigureS7 ), thus confirming that both p-hiPSC lines are derived from HNF cells. Both p-hiPSC lines exhibited the same karyotype as the starting HNF cells (FigureS8 ).
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Figure2
Characterization of p-hiPSC Lines
(A) Quantitative RT-PCR was performed to assess the expression of c-Myc, Gdf-3, Klf4, Nanog, Oct4, Rex1, Sox2, and hTERT in p-hiPS01 and p-hiPS02, hES (H9), and HNF cells. Relative gene expression represents fold changes relative to that of HNF cells normalized to -actin expression. This experiment (repeated twice in triplicate using independently prepared cDNAs) resulted in almost identical patterns.
(B) The global gene-expression patterns were compared between p-hiPS01 and HNF, and between p-hiPS01 and H9 with Affymetrix microarrays. The red lines indicate the diagonal and 5-fold changes between the paired samples.
(C) Bisulfite sequencing analysis of the Nanog and Oct4 promoters reveals almost complete epigenetic reprogramming. Open and closed circles indicate unmethylated and methylated CpG, respectively. Numbers on top show each CpG location. Percentages of CpG methylation (%Me) are shown.
(D) In vitro differentiation of p-hiPSCs. Immunostaining images (first and second row panels) show all three germ layer cells at day 24, including neural (ectodermal), muscle and endothelial-like (mesodermal), and endoderm-like cells (endoderm).
(E) Teratoma formation in immunodeficiency mice by p-hiPSCs. H&E staining was performed for teratomas. The resulting teratomas contained tissues representing all three germ layers (p-hiPS01, first row; and p-hiPS02, second row): ectoderm, epidermal and neural tissue (rosette); mesoderm, bone and cartilage; and endoderm, respiratory epithelium and intestinal-like epithelium.
When the p-hiPSCs were allowed to form embryoid bodies (EBs) by suspension culture, they readily differentiated into cells of all three germ layers (Figure2D, FigureS8 ). After 8 days, well-formed EB structures were observed from both p-hiPSC clones. When these EB-like structures were incubated on gelatin-coated tissue culture plates in ITSFn media for 1525 days, they differentiated to a wide range of cell types, including neural, muscle, and endodermal cells, among others. Immunocytochemical analyses demonstrated the existence of different cell types positive for hepatocyte necrosis factor 3 (HNF 3, endoderm marker), -fetoprotein (AFP, endoderm marker), smooth-muscle actin (SMA, mesoderm marker), desmin (mesoderm marker), Tuj1 (ectoderm marker), nestin (ectoderm marker), and tyrosine hydroxylase (TH, ectoderm marker) (Figure2D, FigureS8 ). In addition, teratoma formation was observed after transplantation of p-hiPSCs under the kidney capsule of nude mice for 68 weeks. These teratomas contained tissues from all three germ layers including neural tissues (ectoderm), epidermal tissues (ectoderm), striated muscle (mesoderm), adipose tissue (mesoderm), cartilage (mesoderm), respiratory epithelium (endoderm), and intestinal-like epithelial tissues (endoderm) (Figure2E, FigureS8 ), confirming that both p-hiPSC clones exhibit pluripotency both invitro and invivo.
Protein-based hiPSC technology offers a new and potentially safe method for generating patient-specific stem cells that does not require the destruction of ex utero embryos. This system completely eliminates genome manipulation and DNA transfection, resulting in human iPSCs suitable for drug discovery, disease modeling, and future clinical translation. In this regard, the present study demonstrates the proof of concept that human iPSCs can be generated by direct protein delivery without genetic manipulation. Other studies suggest that it may be possible to replace and/or further reduce the number of factors required for reprogramming (Huangfu etal., 2008,Li etal., 2009,Shi etal., 2008). To minimize/avoid chromosomal disruption, adenovirus and plasmid transfection have been successfully used to generate iPSCs in the mouse system (Kaji etal., 2009,Okita etal., 2008). Also, Thomson and his colleagues reported generation of hiPSCs by transfection with nonintegrating episomal vectors (Yu etal., 2009). In addition, piggyBac transposon (Kaji etal., 2009,Woltjen etal., 2009) and Cre-recombinase excisable viruses (Soldner etal., 2009) have been used to generate hiPSCs. While the transgenes can be excised by inducible gene expression once reprogramming is established (Soldner etal., 2009,Stadtfeld etal., 2008,Woltjen etal., 2009), residual sequences and chromosomal disruptions may still result in harmful alterations that could pose clinical risks.
The DNA vector-free, direct protein transduction system described here eliminates limitations that may be caused by viral or any other DNA-based reprogramming methods. However, the generation of p-hiPSCs is very slow and inefficient and requires further optimization. In particular, the whole-protein extracts used in the present study limited the concentrations of factors delivered into the target cells, thus suggesting that p-hiPSCs may be more efficiently generated using purified reprogramming proteins. Recently, Ding and his colleagues reported the generation of mouse iPSCs by combining the use of recombinant reprogramming proteins and the small molecule valproic acid (Zhou etal., 2009). In this study, mouse iPSCs were not generated when only recombinant proteins were used. In contrast, the system described here generated human iPSCs with direct delivery of reprogramming proteins in the absence of any chemical treatment. One possible explanation for these differences is that we used reprogramming proteins expressed in mammalian cells, while Ding and colleagues used refolded proteins after expression in E. coli. Since chemicals such as valproic acid and/or genetic manipulation may induce mutations, it has been suggested that whole genomic sequencing would be necessary if such methods are used to generate iPSCs (Yamanaka, 2009). In conclusion, the system described here eliminates the potential risks associated with chromosomal integrations and/or mutations and may allow the translation of hiPSC technology into the clinic.
Acknowledgments
This work was supported by National Institutes of Health (NIH) grants MH48866 and DC 006501 and by International Grants from the CHA University, Korean Stem Cell Research Center, and Dongyang Corporation Co. in Korea. The authors thank Dr. V. Morgan (Harvard Partners Center for Genetics and Genomics), Dr. J. Kim (Harvard Medical School), and Dr. J. Lee (Shippensburg University) for microarray analysis and Ms. J. Johnson (Cell Line Genetics) for karyotyping analysis. R.L. is an employee and shareholder of Advanced Cell Technology and a scientific advisor for Stem Cell and Regenerative Medicine International. K.Y.C. is a shareholder of CHA Bio and Diostech Co., Ltd., and Stem Cell and Regenerative Medicine International.
Accession Numbers
Microarray data can be assessed at Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE16093, GSM402806, GSM402752, GSM402717, GSM402708, and GSM402707.
Supplemental Data
Document S1. Supplemental Experimental Procedures, Supplemental References, Eight Figures, and Four Tables (PDF 497 kb)
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Publication Information
Received: May 6, 2009
Revised: May 11, 2009
Accepted: May 12, 2009
Published online: May 28, 2009
If Anyone has some time. Please send these people an email and tell them about the news we all learned on May 28th 09.
http://www.catholic.net/index.php?option=contact
how does this trade at 20 cents? who buys this stock? I am always interested in looking at the Pump and Dump stocks after they exhale, very suprisd this is not subpenny as of yet.
How many Alias' do you have?
Not until a massive reverse split. nothing will happen til Jan 2010 when the company needs money again. Pumped and then Dumped.
below should answer your question. From the below, we will not only have the financials by the end of this month/June, but be listed once more on the BB...
Advanced Cell Technology, Inc. (Advanced Cell, ACT) (OTC: ACTC) announced today that it has filed its Form 10-Q for its 2008 second and third quarters for the three months ended June 30, 2008 and September 30, 2008. The Company anticipates filing its Form 10-K for the year-ended December 31, 2008 and Form10-Q for the first quarter ended March 31, 2009 prior to the end of this quarter. Upon completing those filings, the Company intends to file for relisting on the Over-the-Counter Bulletin Board as its financial statements will be current.
“We recognize the importance of transparency and the need to become current in our financial reporting and are working diligently with our auditors to achieve this,” said William Caldwell, CEO of Advanced Cell. “Filing the 10-Q’s for two quarters last year is the first step in the process that will hopefully lead to a listing on the Bulletin Board. We believe such a listing should increase interest among the investment community in the significant progress we have made in developing our scientific platform, and our IND filing later this year when we will seek approval to commence human clinical trials for our retinal pigment epithelium (RPE) technology.”
http://ih.advfn.com/p.php?pid=nmona&cb=1243877044&article=37664322&symbol=NO%5EACTC
It makes sense financially for Advanced to be located in worcester, building space is far less expensive than Route 9, or Suburbs of Boston. The memory of previous doom/gloom financially for Advanced will keep them grounded in Worcester or a location where rents/terms are more favorable.
Yeah maybe?
but the Creditors have a set allotement of shares they sell every day.. This PPS can not run more than 20% {yet] with this selling pressure..???.
LOOK EVERYONE A UL LISTING!!!!!!!!!!
15 MILLION bulbs to be shipped in the next 6 months!!!!!!!!!!!!!!!!!!! oooooo how sweet it is to see the shorts cover!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
Amex listing in the next 2-3 weeks and or before July 15, 2009 a PPS of 2.15!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
It is a redeemable guarantee that can be converted to common shares. It is in 1st position before preferred and common.
REVENUE!!!!!!!!!!! shorts cover and Longs rejoice!!!!!!!!!!!