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1.
Cytotherapy ; 2024 May 31.
Article in English | MEDLINE | ID: mdl-38934983

ABSTRACT

BACKGROUND AIMS: With the continuous development and advancement of human pluripotent stem cell (PSC)-derived cell therapies, an ever-increasing number of clinical indications can benefit from their application. Due to the capacity for PSCs to form teratomas, safety testing is required to ensure the absence of residual PSCs in a cell product. To mitigate these limitations, in vitro analytical methods can be utilized as quality control after the production of a PSC-derived cell product. Sensitivity of these analytic methods is critical in accurately quantifying residual PSC in the final cell product. In this study, we compared the sensitivity of three in vitro assays: qPCR, ddPCR and RT-LAMP. METHODS: The spike-in samples were produced from three independent experiments, each spiked with different PSC lines (PSC1, NH50191, and WA09 referred to as H9) into a background of primary fibroblasts (Hs68). These samples were then subjected to qPCR, ddPCR and RT-LAMP to determine their detection limit in measuring a commonly used PSC marker, LIN28A. RESULTS: The results indicated that the three analytic methods all exhibited consistent results across different cell-line spiked samples, with ddPCR demonstrating the highest sensitivity of the three methods. The LIN28A ddPCR assay could confidently detect 10 residual PSCs in a million fibroblasts. DISCUSSION: In our hand, ddPCR LIN28A assay demonstrated the highest sensitivity for detection of residual PSCs compared to the other two assays. Correlating such in vitro safety results with corresponding in vivo studies demonstrating the tumorigenicity profile of PSC-derived cell therapy could accelerate the safe clinical translation of cell therapy.

2.
Oncotarget ; 6(26): 22258-69, 2015 Sep 08.
Article in English | MEDLINE | ID: mdl-26082437

ABSTRACT

Studies have implicated that a small sub-population of cells within a tumour, termed cancer stem cells (CSCs), have an enhanced capacity for tumour formation in multiple cancers and may be responsible for recurrence of the disease after treatment, including radiation. Although comparisons have been made between CSCs and bulk-tumour, the more important comparison with respect to therapy is between tumour-sustaining CSC versus normal stem cells that maintain the healthy tissue. However, the absence of normal known counterparts for many CSCs has made it difficult to compare the radiation responses of CSCs with the normal stem cells required for post-radiotherapy tissue regeneration and the maintenance of tissue homeostasis. Here we demonstrate that transformed human embryonic stem cells (t-hESCs), showing features of neoplastic progression produce tumours resistant to radiation relative to their normal counterpart upon injection into immune compromised mice. We reveal that t-hESCs have a reduced capacity for radiation induced cell death via apoptosis and exhibit altered cell cycle arrest relative to hESCs in vitro. t-hESCs have an increased expression of BclXL in comparison to their normal counterparts and re-sensitization of t-hESCs to radiation upon addition of BH3-only mimetic ABT737, suggesting that overexpression of BclXL underpins t-hESC radiation insensitivity. Using this novel discovery platform to investigate radiation resistance in human CSCs, our study indicates that chemotherapy targeting Bcl2-family members may prove to be an adjuvant to radiotherapy capable of targeting CSCs.


Subject(s)
Human Embryonic Stem Cells/radiation effects , Neoplasms/radiotherapy , Neoplastic Stem Cells/radiation effects , Animals , Cell Differentiation/radiation effects , Cell Proliferation/radiation effects , Cells, Cultured , Heterografts , Human Embryonic Stem Cells/pathology , Humans , Mice , Mice, Inbred NOD , Mice, Transgenic , Neoplastic Stem Cells/pathology
3.
Stem Cells ; 33(6): 1839-49, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25764124

ABSTRACT

Current treatments that use hematopoietic progenitor cell (HPC) transplantation in acute myeloid leukemia (AML) patients substantially reduce the risk of relapse, but are limited by the availability of immune compatible healthy HPCs. Although cellular reprogramming has the potential to provide a novel autologous source of HPCs for transplantation, the applicability of this technology toward the derivation of healthy autologous hematopoietic cells devoid of patient-specific leukemic aberrations from AML patients must first be evaluated. Here, we report the generation of human AML patient-specific hematopoietic progenitors that are capable of normal in vitro differentiation to myeloid lineages and are devoid of leukemia-associated aberration found in matched patient bone marrow. Skin fibroblasts were obtained from AML patients whose leukemic cells possessed a distinct, leukemia-associated aberration, and used to create AML patient-specific induced pluripotent stem cells (iPSCs). Through hematopoietic differentiation of AML patient iPSCs, coupled with cytogenetic interrogation, we reveal that AML patient-specific HPCs possess normal progenitor capacity and are devoid of leukemia-associated mutations. Importantly, in rare patient skin samples that give rise to mosaic fibroblast cultures that continue to carry leukemia-associated mutations; healthy hematopoietic progenitors can also be generated via reprogramming selection. Our findings provide the proof of principle that cellular reprogramming can be applied on a personalized basis to generate healthy HPCs from AML patients, and should further motivate advances toward creating transplantable hematopoietic stem cells for autologous AML therapy.


Subject(s)
Cell Differentiation/genetics , Cellular Reprogramming/genetics , Genome, Human , Hematopoietic Stem Cells/cytology , Induced Pluripotent Stem Cells/cytology , Leukemia, Myeloid, Acute/therapy , Bone Marrow/immunology , Cell Differentiation/physiology , Hematopoietic Stem Cell Transplantation , Humans , Leukemia, Myeloid, Acute/genetics , Neoplastic Stem Cells/cytology
4.
Blood ; 117(26): 7112-20, 2011 Jun 30.
Article in English | MEDLINE | ID: mdl-21562049

ABSTRACT

Xenotransplantation of acute myeloid leukemia (AML) into immunodeficient mice has been critical for understanding leukemogenesis in vivo and defining self-renewing leukemia-initiating cell subfractions (LICs). Although AML-engraftment capacity is considered an inherent property of LICs, substrains of NOD/SCID mice that possess additional deletions such as the IL2Rγc(null) (NSG) have been described as a more sensitive recipient to assay human LIC function. Using 23 AML-patient samples, 39% demonstrated no detectable engraftment in NOD/SCID and were categorized as AMLs devoid of LICs. However, 33% of AML patients lacking AML-LICs were capable of engrafting NSG recipients, but produced a monoclonal T-cell proliferative disorder similar to T-ALL. These grafts demonstrated self-renewal capacity as measured by in vivo serial passage and were restricted to CD34-positive fraction, and were defined as LICs. Molecular analysis for translocations in MLL genes indicated that these AML patient-derived LICs all expressed the MLL-AFX1 fusion product. Our results reveal that the in vivo human versus xenograft host microenvironment dictates the developmental capacity of human LICs residing in a small subset of patients diagnosed with AML harboring MLL mutations. These findings have implications both for the basic biology of CSC function, and for the use of in vivo models of the leukemogenic process in preclinical or diagnostic studies.


Subject(s)
Leukemia, Myeloid, Acute/pathology , Leukemia, T-Cell/pathology , Neoplastic Stem Cells/pathology , Animals , Antigens, CD34/metabolism , Cells, Cultured , Clone Cells/metabolism , Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor , Graft Survival , Histone-Lysine N-Methyltransferase , Humans , Interleukin Receptor Common gamma Subunit/genetics , Interleukin Receptor Common gamma Subunit/metabolism , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, T-Cell/genetics , Leukemia, T-Cell/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Mutant Proteins/genetics , Mutant Proteins/metabolism , Myeloid-Lymphoid Leukemia Protein/genetics , Myeloid-Lymphoid Leukemia Protein/metabolism , Neoplasm Transplantation , Neoplastic Stem Cells/metabolism , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , RNA, Messenger/metabolism , Translocation, Genetic , beta 2-Microglobulin/genetics , beta 2-Microglobulin/metabolism
5.
Nat Biotechnol ; 27(1): 91-7, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19122652

ABSTRACT

Cultured human embryonic stem (hES) cells can acquire genetic and epigenetic changes that make them vulnerable to transformation. As hES cells with cancer-cell characteristics share properties with normal hES cells, such as self-renewal, teratoma formation and the expression of pluripotency markers, they may be misconstrued as superior hES cells with enhanced 'stemness'. We characterize two variant hES cell lines (v-hESC-1 and v-hESC-2) that express pluripotency markers at high levels and do not harbor chromosomal abnormalities by standard cytogenetic measures. We show that the two lines possess some features of neoplastic progression, including a high proliferative capacity, growth-factor independence, a 9- to 20-fold increase in frequency of tumor-initiating cells, niche independence and aberrant lineage specification, although they are not malignant. Array comparative genomic hybridization reveals an amplification at 20q11.1-11.2 in v-hESC-1 and a deletion at 5q34a-5q34b;5q3 and a mosaic gain of chromosome 12 in v-hESC-2. These results emphasize the need for functional characterization to distinguish partially transformed and normal hES cells.


Subject(s)
Embryonic Stem Cells/cytology , Neoplasms/pathology , Cell Differentiation , Cell Line , Cell Line, Tumor , Chromosome Aberrations , Comparative Genomic Hybridization , Cytogenetics , Disease Progression , Fibroblast Growth Factor 2/metabolism , Genetic Techniques , Humans , Nucleic Acid Hybridization , Phenotype , Stem Cells/metabolism
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