Ignite Creation Date:
2024-05-05 @ 4:56 PM
Last Modification Date:
2024-10-26 @ 9:26 AM
Study NCT ID:
NCT00353197
Status:
RECRUITING
Last Update Posted:
2024-03-01
First Post:
2006-07-16
Brief Title:
Derivation of New Human Embryonic Stem Cell Lines Lines for Clinical Use
Sponsor:
Hadassah Medical Organization
Organization:
Hadassah Medical Organization
Study Overview
Official Title:
The Derivation of New Human Embryonic Stem Cell Lines for Clinical Use
Status:
RECRUITING
Status Verified Date:
2024-02
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
Human embryonic stem cells hESCs are isolated from the early human embryo and have the capability to proliferate indefinitely in culture and to develop into nearly every cell of the human body Therefore hESCs may serve as a renewable unlimited source of cells for transplantation therapy Because of the use of animal products in their derivation and due to the lack of appropriate quality and process controls in the manufacturing of existing cell lines worldwide existing hESC lines are not suitable for utilization in transplantation therapy
Our objective is to derive several new hESC lines that will be suitable for clinical trials The investigators plan on deriving the new hESC lines utilizing only FDA-approved raw materials in a non-animal culture system They will be produced entirely under GMP conditions using appropriately documented procedures and analytical methods completely safety tested and screened for infectious and adventitious agents
Our objective is to derive several new hESC lines that will be suitable for clinical trials The investigators plan on deriving the new hESC lines utilizing only FDA-approved raw materials in a non-animal culture system They will be produced entirely under GMP conditions using appropriately documented procedures and analytical methods completely safety tested and screened for infectious and adventitious agents
Detailed Description:
Embryonic stem ES cell lines are derived from the pluripotent cells of the early embryo The key properties of ES cells are their ability to proliferate indefinitely in vitro without differentiation and their capability to give rise to all cells of the body The recent derivation of ES cell lines from human embryos Thomson et al 1998 Reubinoff et al 2000 attracts significant attention due to the remarkable potential of these cells for basic scientific research drug development and regenerative medicine
Since human embryonic stem cells hESCs are capable of unlimited self-renewal and can give rise to any specialized cell via differentiation they can potentially be used as an unlimited donor source of cells for transplantation in a variety of disorders that result from the loss of cells or cellular dysfunction Among these conditions is Parkinsons disease multiple sclerosis traumatic spinal cord injury diabetes heart failure liver failure etc
It has been proposed that the essential characteristics of primate ES cells should include i derivation from the preimplantation or periimplantation embryo ii prolonged undifferentiated proliferation and iii stable developmental potential to form derivatives of all three embryonic germ layers even after prolonged culture Thomson 1996 The investigators have derived stem cell lines from early human embryos that meet these criteria Reubinoff et al 2000 Our group was second in the world to derive hESC lines and the first to show their potential to undergo somatic differentiation in vitro Reubinoff et al 2000
To exploit the remarkable potential of hESCs improvement of currently used methods for culturing and manipulating the cells as well as controlling their differentiation are required In this context the investigators have developed novel approaches to cryopreserve Reubinoff et al 2001 to genetically modify Gropp et al 2003 Ben-Dor et al 2006 and to control the differentiation of hESCs cells Reubinoff et al 2001 Itsykson et al 2005
Human ES cell lines are derived from embryos produced by in vitro fertilization IVF for clinical purposes Surplus frozen embryos that are no longer required for infertility treatment are recruited for this purpose after donor informed consent and institutionalnational review board approvals are obtained The embryos are thawed and cultured to the blastocyst stage 5-6 days the inner cell mass ICM comprised of pluripotent cells is isolated and the stem cells are most commonly cultured on mouse embryonic fibroblast feeder cell layer The feeder layer is required to prevent differentiation and to promote the proliferation of the stem cells
Most hESC lines reported worldwide to date and all the cell lines currently listed in the NIH registry were derived on mouse fibroblast feeder layers The current lines would not be suitable for clinical use as the screening of donors and reagents was not FDA compliant and the presence of the mouse feeders renders these lines xenotransplantation products The regulatory compliance issues these lines raise make it difficult to obtain FDA approval for human transplantation use
To eliminate the use of mouse feeders undifferentiated hESCs can be successfully propagated on laminin or Matrigel-coated plastic surfaces in the presence of mouse embryonic fibroblast-conditioned medium Xu et al 2001 While this system avoids direct contact between mouse feeders and hESCs the risk of cross-transfer of animal pathogens from the animal-conditioned medium to the hESCs is not avoided During the early days of developing our cell lines the investigators successfully cultivated the hESCs on human feeders Subsequently Richards and his colleges demonstrated that human fetal and adult feeders support prolonged undifferentiated propagation of existing hESC lines and are superior to cell-free matrices supplemented with human or mouse feeder-conditioned medium They also reported the derivation of a new hESC line using human embryonic fibroblasts and animal-free culture conditions Richards et al 2002 The potential use of human placenta and foreskin-derived feeders to develop and support undifferentiated propagation of hESCs was also demonstrated Genbacev et al 2005 Amit et al 2003
Maintenance of cultures of undifferentiated hESCs in the absence of feeders was recently reported It was shown that the combination of LIF basic fibroblast growth factor and transforming growth factor Beta1 can support undifferentiated proliferation of hESCs on fibronectin Amit et al 2004 The activation of the Wnt signaling system was also suggested to promote the maintenance of pluripotent hESCs in the absence of feeders Sato et al 2004 Furthermore it has been suggested that substituting the medium with high concentrations of FGF2 and noggin is sufficient to support the propagation of hESCs without feeders Xu et al 2005 In addition feeder-free undifferentiated propagation of hESCs colonies was reported with a chemically-defined medium without serum replacer supplemented with activin or nodal plus FGF2 Vallier er al 2005 Lastly derivation and propagation of hESCs in an animal free chemically-defined feeder-free culture system in the presence of high concentrations of FGF2 was reported Ludwig et al 2006 So far the development of hESC under conditions that will allow there future use for transplantation therapy was not reported
In the second half of the project the investigators propose to continue our efforts to develop new clinical-grade hESC lines derived totally from FDA-approved raw materials in a non-animal culture system
The mouse feeders will be replaced with primary cell lines of human fibroblasts In the first part of the project the investigators have completed the development of master cell banks MCBs of clinical-grade human fibroblast feeders These feeders were produced entirely under GMP conditions within the Hadassah Vector Production Facility using animal-free FDA-approved raw materials for details see Results section To the best of our knowledge the development of GMP clinical-grade human feeders was not reported by other groups
In parallel the investigators have modified the methods for the derivation and culture of hESCs The investigators have developed novel animal-free methods for the isolation of the pluripotent stem cells from human IVF embryos and successfully used these methods in the derivation of new hESC lines The replacement of animal products and FDA non-approved materials with humanized or recombinant clinical-grade materials in the hESC culture system is near completion Validation of the efficacy of the modified culture system is ongoing Preliminary results suggest that the modified culture system can efficiently support undifferentiated cultivation of hESCs
The newly-derived clinical-grade hESC lines will be derived using embryos from couples entirely screened according to organ donation and blood donation guidelines and regulations per the FDA proposed rule for donor suitability In the first year the investigators have completed the recruitment of 14 embryos from three couples Donor medical histories were reviewed and screening blood and swab test results were obtained and documented Samples of donor couple blood were archived for retrospective testing in a locked storage facility Recruitment of additional embryos is ongoing
Upon completion of the characterization and safety testing of the new human feeders expanding them into working lots and validation of the efficacy of the clinical grade-culture system for hESCs the investigators will develop the new hESC lines in the Hadassah University Hospital Vector Production Facility whereby stringent quality control and environmental testing will be maintained All work will be accomplished utilizing only raw materials approved by our Quality Assurance Program provided by vendors who underwent a strict vendor approvalreview process The production work will be performed using techniques adhering to good manufacturing practices GMPs and good tissue practices GTPs
Early passage hESCs and feeders Master Cell Bank will be submitted for phenotypic and latent agent testing endotoxin analysis and testing for viral and adventitious agent contamination The Working Cell Bank of both feeders and hESCs will be also tested for phenotypic characterization reproducibility sterility and mycoplasma detection of latent agents and endotoxins The final product will be retested as per the Master Cell Bank above
By the conclusion of our manufacturing and testing process Hadassahs Stem Cell Program will provide a generic commercial product The procedures and methods that the investigators have developed will be instrumental tools to other stem cell researchers thus paving the way for the future of stem cell research in Israel and worldwide The commercial potential of the product will allow production of specific cells for clinical use they will have a widespread commercial value and will be of enormous market worth
Since human embryonic stem cells hESCs are capable of unlimited self-renewal and can give rise to any specialized cell via differentiation they can potentially be used as an unlimited donor source of cells for transplantation in a variety of disorders that result from the loss of cells or cellular dysfunction Among these conditions is Parkinsons disease multiple sclerosis traumatic spinal cord injury diabetes heart failure liver failure etc
It has been proposed that the essential characteristics of primate ES cells should include i derivation from the preimplantation or periimplantation embryo ii prolonged undifferentiated proliferation and iii stable developmental potential to form derivatives of all three embryonic germ layers even after prolonged culture Thomson 1996 The investigators have derived stem cell lines from early human embryos that meet these criteria Reubinoff et al 2000 Our group was second in the world to derive hESC lines and the first to show their potential to undergo somatic differentiation in vitro Reubinoff et al 2000
To exploit the remarkable potential of hESCs improvement of currently used methods for culturing and manipulating the cells as well as controlling their differentiation are required In this context the investigators have developed novel approaches to cryopreserve Reubinoff et al 2001 to genetically modify Gropp et al 2003 Ben-Dor et al 2006 and to control the differentiation of hESCs cells Reubinoff et al 2001 Itsykson et al 2005
Human ES cell lines are derived from embryos produced by in vitro fertilization IVF for clinical purposes Surplus frozen embryos that are no longer required for infertility treatment are recruited for this purpose after donor informed consent and institutionalnational review board approvals are obtained The embryos are thawed and cultured to the blastocyst stage 5-6 days the inner cell mass ICM comprised of pluripotent cells is isolated and the stem cells are most commonly cultured on mouse embryonic fibroblast feeder cell layer The feeder layer is required to prevent differentiation and to promote the proliferation of the stem cells
Most hESC lines reported worldwide to date and all the cell lines currently listed in the NIH registry were derived on mouse fibroblast feeder layers The current lines would not be suitable for clinical use as the screening of donors and reagents was not FDA compliant and the presence of the mouse feeders renders these lines xenotransplantation products The regulatory compliance issues these lines raise make it difficult to obtain FDA approval for human transplantation use
To eliminate the use of mouse feeders undifferentiated hESCs can be successfully propagated on laminin or Matrigel-coated plastic surfaces in the presence of mouse embryonic fibroblast-conditioned medium Xu et al 2001 While this system avoids direct contact between mouse feeders and hESCs the risk of cross-transfer of animal pathogens from the animal-conditioned medium to the hESCs is not avoided During the early days of developing our cell lines the investigators successfully cultivated the hESCs on human feeders Subsequently Richards and his colleges demonstrated that human fetal and adult feeders support prolonged undifferentiated propagation of existing hESC lines and are superior to cell-free matrices supplemented with human or mouse feeder-conditioned medium They also reported the derivation of a new hESC line using human embryonic fibroblasts and animal-free culture conditions Richards et al 2002 The potential use of human placenta and foreskin-derived feeders to develop and support undifferentiated propagation of hESCs was also demonstrated Genbacev et al 2005 Amit et al 2003
Maintenance of cultures of undifferentiated hESCs in the absence of feeders was recently reported It was shown that the combination of LIF basic fibroblast growth factor and transforming growth factor Beta1 can support undifferentiated proliferation of hESCs on fibronectin Amit et al 2004 The activation of the Wnt signaling system was also suggested to promote the maintenance of pluripotent hESCs in the absence of feeders Sato et al 2004 Furthermore it has been suggested that substituting the medium with high concentrations of FGF2 and noggin is sufficient to support the propagation of hESCs without feeders Xu et al 2005 In addition feeder-free undifferentiated propagation of hESCs colonies was reported with a chemically-defined medium without serum replacer supplemented with activin or nodal plus FGF2 Vallier er al 2005 Lastly derivation and propagation of hESCs in an animal free chemically-defined feeder-free culture system in the presence of high concentrations of FGF2 was reported Ludwig et al 2006 So far the development of hESC under conditions that will allow there future use for transplantation therapy was not reported
In the second half of the project the investigators propose to continue our efforts to develop new clinical-grade hESC lines derived totally from FDA-approved raw materials in a non-animal culture system
The mouse feeders will be replaced with primary cell lines of human fibroblasts In the first part of the project the investigators have completed the development of master cell banks MCBs of clinical-grade human fibroblast feeders These feeders were produced entirely under GMP conditions within the Hadassah Vector Production Facility using animal-free FDA-approved raw materials for details see Results section To the best of our knowledge the development of GMP clinical-grade human feeders was not reported by other groups
In parallel the investigators have modified the methods for the derivation and culture of hESCs The investigators have developed novel animal-free methods for the isolation of the pluripotent stem cells from human IVF embryos and successfully used these methods in the derivation of new hESC lines The replacement of animal products and FDA non-approved materials with humanized or recombinant clinical-grade materials in the hESC culture system is near completion Validation of the efficacy of the modified culture system is ongoing Preliminary results suggest that the modified culture system can efficiently support undifferentiated cultivation of hESCs
The newly-derived clinical-grade hESC lines will be derived using embryos from couples entirely screened according to organ donation and blood donation guidelines and regulations per the FDA proposed rule for donor suitability In the first year the investigators have completed the recruitment of 14 embryos from three couples Donor medical histories were reviewed and screening blood and swab test results were obtained and documented Samples of donor couple blood were archived for retrospective testing in a locked storage facility Recruitment of additional embryos is ongoing
Upon completion of the characterization and safety testing of the new human feeders expanding them into working lots and validation of the efficacy of the clinical grade-culture system for hESCs the investigators will develop the new hESC lines in the Hadassah University Hospital Vector Production Facility whereby stringent quality control and environmental testing will be maintained All work will be accomplished utilizing only raw materials approved by our Quality Assurance Program provided by vendors who underwent a strict vendor approvalreview process The production work will be performed using techniques adhering to good manufacturing practices GMPs and good tissue practices GTPs
Early passage hESCs and feeders Master Cell Bank will be submitted for phenotypic and latent agent testing endotoxin analysis and testing for viral and adventitious agent contamination The Working Cell Bank of both feeders and hESCs will be also tested for phenotypic characterization reproducibility sterility and mycoplasma detection of latent agents and endotoxins The final product will be retested as per the Master Cell Bank above
By the conclusion of our manufacturing and testing process Hadassahs Stem Cell Program will provide a generic commercial product The procedures and methods that the investigators have developed will be instrumental tools to other stem cell researchers thus paving the way for the future of stem cell research in Israel and worldwide The commercial potential of the product will allow production of specific cells for clinical use they will have a widespread commercial value and will be of enormous market worth
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None