Clinical Grade Human Embryonic Stem Research Articles

Grafts Derived from an α-Synuclein Triplication Patient Mediate Functional Recovery but Develop Disease-Associated Pathology in the 6-OHDA Model of Parkinson's Disease.

Background:Human induced pluripotent stem cells (hiPSCs) have been proposed as an alternative source for cell replacement therapy for Parkinson’s disease (PD) and they provide the option of using the patient’s own cells. A few studies have investigated transplantation of patient-derived dopaminergic (DA) neurons in preclinical models; however, little is known about the long-term integrity and function of grafts derived from patients with PD.Objective:To assess the viability and function of DA neuron grafts derived from a patient hiPSC line with an α-synuclein gene triplication (AST18), using a clinical grade human embryonic stem cell (hESC) line (RC17) as a reference control.Methods:Cells were differentiated into ventral mesencephalic (VM)-patterned DA progenitors using an established GMP protocol. The progenitors were then either terminally differentiated to mature DA neurons in vitro or transplanted into 6-hydroxydopamine (6-OHDA) lesioned rats and their survival, maturation, function, and propensity to develop α-synuclein related pathology, were assessed in vivo.Results:Both cell lines generated functional neurons with DA properties in vitro. AST18-derived VM progenitor cells survived transplantation and matured into neuron-rich grafts similar to the RC17 cells. After 24 weeks, both cell lines produced DA-rich grafts that mediated full functional recovery; however, pathological changes were only observed in grafts derived from the α-synuclein triplication patient line.Conclusion:This data shows proof-of-principle for survival and functional recovery with familial PD patient-derived cells in the 6-OHDA model of PD. However, signs of slowly developing pathology warrants further investigation before use of autologous grafts in patients.

RPE tissue engineering based on human pluripotent stem cells for cell therapy

AbstractIn developed countries, retinal degenerative diseases affecting the Retinal Pigment Epithelium (RPE), including Age‐related Macular Degeneration and inherited retinal diseases such as Retinitis Pigmentosa (RP), are the predominant causes of human blindness. Despite the scientific advances achieved in the last years, there is no cure for the majority of patients. In this context, cell therapy based on human pluripotent stem cells is an attractive therapeutic alternative to replace dead or defective RPE cells. Different formulations were proposed: either as a cell suspension or as an engineered tissue. We will discuss the preclinical data obtained with these two strategies. In particular, we will present the preclinical development of our own RPE tissue therapy. This cell therapy consists in RPE cells derived from clinical grade human embryonic stem cells disposed on a biocompatible substrate allowing the formation of a 3D functional sheet, suitable for transplantation. Functionality and safety were evaluated in rodents and the surgical approach in non‐human primates. This therapy is currently evaluated in a phase I/II clinical trial targeting RP patients with mutations in RPE genes.

Derivation of the clinical grade human embryonic stem cell line RCe015-A (RC-11)

The human embryonic stem cell line RCe015-A (RC-11) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a fragmented cleavage stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe015-A (RC-11) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

Derivation of the clinical grade human embryonic stem cell line RCe021-A (RC-17)

The human embryonic stem cell line RCe021-A (RC-17) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a day 3 embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe021-A (RC-17) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

Derivation of the clinical grade human embryonic stem cell line RCe016-A (RC-12)

The human embryonic stem cell line RCe016-A (RC-12) was derived under quality assured compliance with UK regulations, EU Directives and International guidance for tissue procurement, processing and storage according to good manufacturing practice (GMP) standards. The cell line was derived from a cryopreserved blastocyst stage embryo voluntarily donated as surplus to fertility requirements following informed consent. RCe016-A (RC-12) shows normal pluripotency marker expression and differentiation to three germ layers in vitro. Karyology revealed a mixed male karyotype at early passage (P15), which resolved as normal 46XY by passage 33. Microsatellite PCR identity, HLA and blood group typing data is available.

Derivation of the clinical grade human embryonic stem cell line RCe019-A (RC-15)

The human embryonic stem cell line RCe019-A (RC-15) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a cleavage stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe019-A (RC-15) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XX/47XX, +8 female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

Generation of KCL033 clinical grade human embryonic stem cell line

The KCL033 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. The line was also validated for sterility and specific and non-specific human pathogens.

Generation of KCL031 clinical grade human embryonic stem cell line

The KCL031 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.

Generation of KCL034 clinical grade human embryonic stem cell line

The KCL034 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. The line was also validated for sterility, specific and non-specific human pathogens.

Generation of KCL039 clinical grade human embryonic stem cell line

The KCL039 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays.

Generation of KCL040 clinical grade human embryonic stem cell line

The KCL040 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays.

Generation of KCL038 clinical grade human embryonic stem cell line

The KCL038 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays.

Generation of KCL037 clinical grade human embryonic stem cell line

The KCL037 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays.

Generation of KCL032 clinical grade human embryonic stem cell line

The KCL032 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays.

Use of discarded oocytes as a source of parthenote blastocysts to generate clinical grade human embryonic stem cell lines

Use of discarded oocytes as a source of parthenote blastocysts to generate clinical grade human embryonic stem cell lines

Mechanical isolation of the inner cell mass is effective in derivation of new human embryonic stem cell lines

For clinical grade human embryonic stem cell (hESC) lines, a robust derivation system without any substances having animal origin would be required. We have gradually improved our hESC derivations. Human skin fibroblasts were used as feeder cells in derivation of all our 25 permanent fully characterized hESC lines. In the first four derivations, fetal calf serum was used as a supplement in the medium, thereafter, serum replacement medium was used. Immunosurgery generally used for isolation of the inner cell mass (ICM) still involves animal serum and complement. We developed a practical mechanical isolation method for the ICM. Two flexible metal needles with sharpened tips, 0.125 mm in diameter, were used to open the zona pellucida and extract the ICM under a stereomicroscope. Immunohistochemical and karyotype characterization of the new hESC lines was carried out, and pluripotency was tested in vitro (immunocytochemistry and RT-PCR) and in vivo (teratoma growth). Five hESC lines were obtained from 19 supernumerary blastocysts collected in 2005-2006 (26%), whereas in similar conditions, we obtained 16 lines from 100 blastocysts (16%) using immunosurgery in 2003-2005. The new lines had a normal karyotype and tissues originating from the three embryonic germ cell layers were present. Mechanical isolation of the ICM proved to be an effective way to derive new hESC lines. The technique is fast, does not require any extra investment and the xeno-components of immunosurgery could be avoided.