Natural killer cells (NK cells) are the first line of the innate immune defense system, primarily... more Natural killer cells (NK cells) are the first line of the innate immune defense system, primarily located in peripheral circulation and lymphoid tissues. They kill virally infected and malignant cells through a balancing play of inhibitory and stimulatory receptors. In pre-clinical investigational studies, NK cells show promising anti-tumor effects and are used in adoptive transfer of activated and expanded cells, ex-vivo. NK cells express co-stimulatory molecules that are attractive targets for the immunotherapy of cancers. Recent clinical trials are investigating the use of CAR-NK for different cancers to determine the efficiency. Herein, we review NK cell therapy approaches (NK cell preparation from tissue sources, ways of expansion ex-vivo for “off-the-shelf” allogeneic cell-doses for therapies, and how different vector delivery systems are used to engineer NK cells with CARs) for cancer immunotherapy.
Human mesenchymal stromal cell (hMSC) therapy has been gaining immense interest in regenerative m... more Human mesenchymal stromal cell (hMSC) therapy has been gaining immense interest in regenerative medicine and quite recently for its immunomodulatory properties in COVID-19 treatment. Currently, the use of hMSCs for various diseases is being investigated in >900 clinical trials. Despite the huge effort, setting up consistent and robust scalable manufacturing to meet regulatory compliance across various global regions remains a nagging challenge. This is in part due to a lack of definitive consensus for quality control checkpoint assays starting from cell isolation to expansion and final release criterion of clinical grade hMSCs. In this review, we highlight the bottlenecks associated with hMSC-based therapies and propose solutions for consistent GMP manufacturing of hMSCs starting from raw materials selection, closed and modular systems of manufacturing, characterization, functional testing, quality control, and safety testing for release criteria. We also discuss the standard reg...
International journal of molecular medicine, Jan 10, 2017
Preclinical studies have suggested that paracrine factors from adipose-derived stem cells (ASCs) ... more Preclinical studies have suggested that paracrine factors from adipose-derived stem cells (ASCs) promote the healing of chronic wounds, and that the exposure of ASCs to hypoxia enhances their wound healing effect. To aid the translation of these findings into clinical use, robust wound models are necessary to explore each aspect of wound healing. The aspect of re-epithelization is often studied in a scratch assay based on transformed keratinocytes. However, there are concerns regarding the validity of this model, since these cell lines differ from normal keratinocytes, both in terms of proliferative capacity and differentiation, and sensitivity to environmental cues. In this study, the main challenge of using primary keratinocytes to examine the effects of ASCs was identified to be their different requirements for calcium in the culture media. We confirmed that a high calcium content led to morphological and cytoskeletal changes in primary keratinocytes, and demonstrated that a low ...
... Cell cycle was modeled using ModFit LT™ (Verity Software) to determine fitted S-phase fractio... more ... Cell cycle was modeled using ModFit LT™ (Verity Software) to determine fitted S-phase fraction (% proliferation) and compared to the percent proliferating cells determined by Click-iT® EdU analysis (data not shown). % proliferation Passage 7 0 10 20 30 40 50 60 70 ...
Lead (Pb 2+) is a toxic heavy metal that has adverse effects on the health of humans and other an... more Lead (Pb 2+) is a toxic heavy metal that has adverse effects on the health of humans and other animals. The developing central nervous system is especially sensitive and vulnerable to Pb 2+ toxicity. In this study, the effects of low levels of Pb 2+ exposure on human SH-SY5Y neuroblastoma cell cultures were assessed. The cells were exposed to Pb 2+ (0.01 µM-10 µM) for 48 hrs, and the level of cell proliferation was determined. Pb 2+ significantly inhibited the proliferation of neuroblastoma cells in a concentration-dependent manner. A 50% inhibition (IC 50) in cellular proliferation was observed with 5 µM Pb 2+. A significant decrease in the levels of glutathione (GSH), a critical intracellular antioxidant, was observed at all the lead concentrations. There was also a multifold increase in the activity of caspase-3, a key executioner of apoptosis, and in the levels of prostaglandin E 2 (PGE 2). Our results suggest that the neurotoxic effects of Pb may be mediated by apoptosis and PGE 2 release, which could be potentially detrimental to neuronal survival.
The potential applications of human embryonic stem cells (hESCs) in regenerative medicine and dev... more The potential applications of human embryonic stem cells (hESCs) in regenerative medicine and developmental research have made stem cell biology one of the most fascinating and rapidly expanding fields of biomedicine. The first clinical trial of hESCs in humans has begun, and the field of stem cell therapy has just entered a new era. Here, we report seven hESC lines (SEES-1,-2,-3,-4,-5,-6, and-7). Four of them were derived and maintained on irradiated human mesenchymal stem cells (hMSCs) grown in xenogeneic-free defined media and substrate. Xenogeneic-free hMSCs isolated from the subcutaneous tissue of extra fingers from individuals with polydactyly showed appropriate potentials as feeder layers in the pluripotency and growth of hESCs. In this report, we describe a comprehensive characterization of these newly derived SEES cell lines. In addition, we developed a scalable culture system for hESCs having high biological safety by using gamma-irradiated serum replacement and pharmaceutical-grade recombinant basic fibroblast growth factor (bFGF, also known as trafermin). This is first report describing the maintenance of hESC pluripotency using pharmaceutical-grade human recombinant bFGF (trafermin) and gamma-irradiated serum replacement. Our defined medium system provides a path to scalability in Good Manufacturing Practice (GMP) settings for the generation of clinically relevant cell types from pluripotent cells for therapeutic applications.
Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced plur... more Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, are unique cell sources for disease modeling, drug discovery screens, and cell therapy applications. The first step in producing neural lineages from hPSCs is the generation of neural stem cells (NSCs). Current methods of NSC derivation involve the time-consuming, laborintensive steps of an embryoid body generation or coculture with stromal cell lines that result in low-efficiency derivation of NSCs. In this study, we report a highly efficient serum-free pluripotent stem cell neural induction medium that can induce hPSCs into primitive NSCs (pNSCs) in 7 days, obviating the need for time-consuming, laborious embryoid body generation or rosette picking. The pNSCs expressed the neural stem cell markers Pax6, Sox1, Sox2, and Nestin; were negative for Oct4; could be expanded for multiple passages; and could be differentiated into neurons, astrocytes, and oligodendrocytes, in addition to the brain region-specific neuronal subtypes GABAergic, dopaminergic, and motor neurons. Global gene expression of the transcripts of pNSCs was comparable to that of rosette-derived and human fetal-derived NSCs. This work demonstrates an efficient method to generate expandable pNSCs, which can be further differentiated into central nervous system neurons and glia with temporal, spatial, and positional cues of brain regional heterogeneity. This method of pNSC derivation sets the stage for the scalable production of clinically relevant neural cells for cell therapy applications in good manufacturing practice conditions.
Human-induced pluripotent stem cells (iPSCs) are an important potential source of cells for regen... more Human-induced pluripotent stem cells (iPSCs) are an important potential source of cells for regenerative medicine due to their inherent ability to differentiate into all cell types of the three germ layers. Generation of iPSCs with a non-integrating reprogramming method and in culture conditions that are completely absent of animal proteins will be ideal for such regenerative and cell therapy applications. Here we describe a method to generate non-integrating iPSCs using the Episomal iPSC Reprogramming Vectors.
Large-scale production of human embryonic stem cells (hESCs) is the most inevitable choice to ful... more Large-scale production of human embryonic stem cells (hESCs) is the most inevitable choice to fulfill biomedical research needs to produce mature, functional, and pure derivatives of cell types that can be utilized for transplantation purposes or in drug ...
Levels of RNA, mRNA and separation of ribosomal proteins from control and ethanol treated rat liv... more Levels of RNA, mRNA and separation of ribosomal proteins from control and ethanol treated rat liver, showed no change in total RNA content, but poly(A+)mRNA was reduced significantly in ethanolic rats. Ribosomal proteins $2, S3a, S3b, $4, L3, L4, L4a, L10a and L15 were found substantially reduced in experimental rat livers. This study suggests decrease in poly(A ÷) mRNA coupled with loss of ribosomal proteins must be responsible for decreased protein synthesis in chronic alcoholism. (Mol Cell Biochem 115: 143-147, 1992)
Oxidative injury is believed to be a major factor in the pathogenesis of a variety of neurodegene... more Oxidative injury is believed to be a major factor in the pathogenesis of a variety of neurodegenerative diseases. Additionally, the mode of cell death in oxidant-stressed cells can vary. The present study was conducted to evaluate the use of a primary neuronal cell-based bioassay in which different modes of oxidant-induced cell death could be studied and in which putative neuroprotective agents could be screened. Addition of 50 AM H 2 O 2 to primary cortical neuronal cultures for 1 h under normal ATP conditions resulted in approximately 40% cell death, almost exclusively of an apoptotic nature. In this condition, cell death was effectively blocked by GM1 ganglioside, the semi-synthetic ganglioside derivative LIGA20, the dopamine receptor agonist pramipexole (PPX) and the caspase inhibitor Z-VAD-FMK but not by the poly (ADP-ribose) polymerase (PARP) inhibitor 3-aminobenzamide (3-AB). Pretreatment of cells with 0.01 AM oligomycin for 45 min prior to addition of 50 AM H 2 O 2 caused significant ATP depletion and approximately the same amount of cell death as H 2 O 2 alone. However, under these conditions, cell death was primarily non-apoptotic in nature and GM1, LIGA20 and Z-VAD-FMK had no protective effects. In contrast, AB and PPX effectively blocked cell death. These results suggest that cellular ATP plays a critical role in determining the mode of cell death in primary neurons and that these types of in vitro models may provide a useful system for screening putative neuroprotective agents.
The large cell doses (&am... more The large cell doses (>1 × 10(6) cells/kg) used in clinical trials with mesenchymal stem/stromal cells (MSC) will require an efficient production process. Moreover, monitoring and control of MSC ex-vivo expansion is critical to provide a safe and reliable cell product. Bioprocess engineering approaches, such as bioreactor technology, offer the adequate tools to develop and optimize a cost-effective culture system for the rapid expansion of human MSC for cellular therapy. Herein, a xenogeneic (xeno)-free microcarrier-based culture system was successfully established for bone marrow (BM) MSC and adipose tissue-derived stem/stromal cell (ASC) cultivation using a 1L-scale controlled stirred-tank bioreactor, allowing the production of (1.1 ± 0.1) × 10(8) and (4.5 ± 0.2) × 10(7) cells for BM MSC and ASC, respectively, after 7 days. Additionally, the effect of different percent air saturation values (%Airsat ) and feeding regime on the proliferation and metabolism of BM MSC was evaluated. No significant differences in cell growth and metabolic patterns were observed under 20% and 9%Airsat . Also, the three different feeding regimes studied-(i) 25% daily medium renewal, (ii) 25% medium renewal every 2 days, and (iii) fed-batch addition of concentrated nutrients and growth factors every 2 days-yielded similar cell numbers, and only slight metabolic differences were observed. Moreover, the immunophenotype (positive for CD73, CD90 and CD105 and negative for CD31, CD80 and HLA-DR) and multilineage differentiative potential of expanded cells were not affected upon bioreactor culture. These results demonstrated the feasibility of expanding human MSC from different sources in a clinically relevant expansion configuration in a controlled microcarrier-based stirred culture system under xeno-free conditions. The further optimization of this bioreactor culture system will represent a crucial step towards an efficient GMP-compliant clinical-scale MSC production system.
The immunomodulatory properties of mesenchymal stem cells (MSCs) make them attractive therapeutic... more The immunomodulatory properties of mesenchymal stem cells (MSCs) make them attractive therapeutic agents for a wide range of diseases. However, the highly demanding cell doses used in MSC clinical trials (up to millions of cells/kg patient) currently require labor intensive methods and incur high reagent costs. Moreover, the use of xenogenic (xeno) serum-containing media represents a risk of contamination and raises safety concerns. Bioreactor systems in combination with novel xeno-free medium formulations represent a viable alternative to reproducibly achieve a safe and reliable MSC doses relevant for cell therapy. The main goal of the present study was to develop a complete xeno-free microcarrier-based culture system for the efficient expansion of human MSC from two different sources, human bone marrow (BM), and adipose tissue. After 14 days of culture in spinner flasks, BM MSC reached a maximum cell density of (2.0 -0.2) • 10 5 cells$mL -1 (18 -1-fold increase), whereas adipose tissue-derived stem cells expanded to (1.4 -0.5) • 10 5 cells$mL -1 (14 -7-fold increase). After the expansion, MSC expressed the characteristic markers CD73, CD90, and CD105, whereas negative for CD80 and human leukocyte antigen (HLA)-DR. Expanded cells maintained the ability to differentiate robustly into osteoblast, adipocyte, and chondroblast lineages upon directed differentiation. These results demonstrated the feasibility of expanding human MSC in a scalable microcarrier-based stirred culture system under xeno-free conditions and represent an important step forward for the implementation of a Good Manufacturing Practicescompliant large-scale production system of MSC for cellular therapy.
DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme composed of a DNA-binding component ... more DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme composed of a DNA-binding component called Ku70/80 and a catalytic subunit called DNA-PKcs. Many investigators have utilized DNA-PKcs-deficient cells and cell lines derived from severe combined immunodeficiency (scid) mice to study DNA repair and apoptosis. However, little is known about the CNS of these mice. This study was carried out using primary neuronal cultures derived from the cerebral hemispheres of new-born wild-type and scid mice to investigate the effects of loss of DNA-PK function on neuronal maturation and survival. Purified neuronal cultures developed comparably in terms of neurite formation and expression of neuronal markers, but scid cultures showed a significant increase in the percentage of dying cells. Furthermore, when apoptosis was induced by staurosporine, scid neurons died more rapidly and in higher numbers. Apoptotic scid neurons exhibited nuclear condensation, DNA fragmentation and caspase-3 activation, but treatment with the general caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl) fluoromethyl ketone did not prevent staurosporine-induced apoptosis. We conclude that a DNA-PK deficiency in cultured scid neurons may cause an accumulation of DNA damage and increased susceptibility to caspase-independent forms of programmed cell death.
Natural killer cells (NK cells) are the first line of the innate immune defense system, primarily... more Natural killer cells (NK cells) are the first line of the innate immune defense system, primarily located in peripheral circulation and lymphoid tissues. They kill virally infected and malignant cells through a balancing play of inhibitory and stimulatory receptors. In pre-clinical investigational studies, NK cells show promising anti-tumor effects and are used in adoptive transfer of activated and expanded cells, ex-vivo. NK cells express co-stimulatory molecules that are attractive targets for the immunotherapy of cancers. Recent clinical trials are investigating the use of CAR-NK for different cancers to determine the efficiency. Herein, we review NK cell therapy approaches (NK cell preparation from tissue sources, ways of expansion ex-vivo for “off-the-shelf” allogeneic cell-doses for therapies, and how different vector delivery systems are used to engineer NK cells with CARs) for cancer immunotherapy.
Human mesenchymal stromal cell (hMSC) therapy has been gaining immense interest in regenerative m... more Human mesenchymal stromal cell (hMSC) therapy has been gaining immense interest in regenerative medicine and quite recently for its immunomodulatory properties in COVID-19 treatment. Currently, the use of hMSCs for various diseases is being investigated in >900 clinical trials. Despite the huge effort, setting up consistent and robust scalable manufacturing to meet regulatory compliance across various global regions remains a nagging challenge. This is in part due to a lack of definitive consensus for quality control checkpoint assays starting from cell isolation to expansion and final release criterion of clinical grade hMSCs. In this review, we highlight the bottlenecks associated with hMSC-based therapies and propose solutions for consistent GMP manufacturing of hMSCs starting from raw materials selection, closed and modular systems of manufacturing, characterization, functional testing, quality control, and safety testing for release criteria. We also discuss the standard reg...
International journal of molecular medicine, Jan 10, 2017
Preclinical studies have suggested that paracrine factors from adipose-derived stem cells (ASCs) ... more Preclinical studies have suggested that paracrine factors from adipose-derived stem cells (ASCs) promote the healing of chronic wounds, and that the exposure of ASCs to hypoxia enhances their wound healing effect. To aid the translation of these findings into clinical use, robust wound models are necessary to explore each aspect of wound healing. The aspect of re-epithelization is often studied in a scratch assay based on transformed keratinocytes. However, there are concerns regarding the validity of this model, since these cell lines differ from normal keratinocytes, both in terms of proliferative capacity and differentiation, and sensitivity to environmental cues. In this study, the main challenge of using primary keratinocytes to examine the effects of ASCs was identified to be their different requirements for calcium in the culture media. We confirmed that a high calcium content led to morphological and cytoskeletal changes in primary keratinocytes, and demonstrated that a low ...
... Cell cycle was modeled using ModFit LT™ (Verity Software) to determine fitted S-phase fractio... more ... Cell cycle was modeled using ModFit LT™ (Verity Software) to determine fitted S-phase fraction (% proliferation) and compared to the percent proliferating cells determined by Click-iT® EdU analysis (data not shown). % proliferation Passage 7 0 10 20 30 40 50 60 70 ...
Lead (Pb 2+) is a toxic heavy metal that has adverse effects on the health of humans and other an... more Lead (Pb 2+) is a toxic heavy metal that has adverse effects on the health of humans and other animals. The developing central nervous system is especially sensitive and vulnerable to Pb 2+ toxicity. In this study, the effects of low levels of Pb 2+ exposure on human SH-SY5Y neuroblastoma cell cultures were assessed. The cells were exposed to Pb 2+ (0.01 µM-10 µM) for 48 hrs, and the level of cell proliferation was determined. Pb 2+ significantly inhibited the proliferation of neuroblastoma cells in a concentration-dependent manner. A 50% inhibition (IC 50) in cellular proliferation was observed with 5 µM Pb 2+. A significant decrease in the levels of glutathione (GSH), a critical intracellular antioxidant, was observed at all the lead concentrations. There was also a multifold increase in the activity of caspase-3, a key executioner of apoptosis, and in the levels of prostaglandin E 2 (PGE 2). Our results suggest that the neurotoxic effects of Pb may be mediated by apoptosis and PGE 2 release, which could be potentially detrimental to neuronal survival.
The potential applications of human embryonic stem cells (hESCs) in regenerative medicine and dev... more The potential applications of human embryonic stem cells (hESCs) in regenerative medicine and developmental research have made stem cell biology one of the most fascinating and rapidly expanding fields of biomedicine. The first clinical trial of hESCs in humans has begun, and the field of stem cell therapy has just entered a new era. Here, we report seven hESC lines (SEES-1,-2,-3,-4,-5,-6, and-7). Four of them were derived and maintained on irradiated human mesenchymal stem cells (hMSCs) grown in xenogeneic-free defined media and substrate. Xenogeneic-free hMSCs isolated from the subcutaneous tissue of extra fingers from individuals with polydactyly showed appropriate potentials as feeder layers in the pluripotency and growth of hESCs. In this report, we describe a comprehensive characterization of these newly derived SEES cell lines. In addition, we developed a scalable culture system for hESCs having high biological safety by using gamma-irradiated serum replacement and pharmaceutical-grade recombinant basic fibroblast growth factor (bFGF, also known as trafermin). This is first report describing the maintenance of hESC pluripotency using pharmaceutical-grade human recombinant bFGF (trafermin) and gamma-irradiated serum replacement. Our defined medium system provides a path to scalability in Good Manufacturing Practice (GMP) settings for the generation of clinically relevant cell types from pluripotent cells for therapeutic applications.
Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced plur... more Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, are unique cell sources for disease modeling, drug discovery screens, and cell therapy applications. The first step in producing neural lineages from hPSCs is the generation of neural stem cells (NSCs). Current methods of NSC derivation involve the time-consuming, laborintensive steps of an embryoid body generation or coculture with stromal cell lines that result in low-efficiency derivation of NSCs. In this study, we report a highly efficient serum-free pluripotent stem cell neural induction medium that can induce hPSCs into primitive NSCs (pNSCs) in 7 days, obviating the need for time-consuming, laborious embryoid body generation or rosette picking. The pNSCs expressed the neural stem cell markers Pax6, Sox1, Sox2, and Nestin; were negative for Oct4; could be expanded for multiple passages; and could be differentiated into neurons, astrocytes, and oligodendrocytes, in addition to the brain region-specific neuronal subtypes GABAergic, dopaminergic, and motor neurons. Global gene expression of the transcripts of pNSCs was comparable to that of rosette-derived and human fetal-derived NSCs. This work demonstrates an efficient method to generate expandable pNSCs, which can be further differentiated into central nervous system neurons and glia with temporal, spatial, and positional cues of brain regional heterogeneity. This method of pNSC derivation sets the stage for the scalable production of clinically relevant neural cells for cell therapy applications in good manufacturing practice conditions.
Human-induced pluripotent stem cells (iPSCs) are an important potential source of cells for regen... more Human-induced pluripotent stem cells (iPSCs) are an important potential source of cells for regenerative medicine due to their inherent ability to differentiate into all cell types of the three germ layers. Generation of iPSCs with a non-integrating reprogramming method and in culture conditions that are completely absent of animal proteins will be ideal for such regenerative and cell therapy applications. Here we describe a method to generate non-integrating iPSCs using the Episomal iPSC Reprogramming Vectors.
Large-scale production of human embryonic stem cells (hESCs) is the most inevitable choice to ful... more Large-scale production of human embryonic stem cells (hESCs) is the most inevitable choice to fulfill biomedical research needs to produce mature, functional, and pure derivatives of cell types that can be utilized for transplantation purposes or in drug ...
Levels of RNA, mRNA and separation of ribosomal proteins from control and ethanol treated rat liv... more Levels of RNA, mRNA and separation of ribosomal proteins from control and ethanol treated rat liver, showed no change in total RNA content, but poly(A+)mRNA was reduced significantly in ethanolic rats. Ribosomal proteins $2, S3a, S3b, $4, L3, L4, L4a, L10a and L15 were found substantially reduced in experimental rat livers. This study suggests decrease in poly(A ÷) mRNA coupled with loss of ribosomal proteins must be responsible for decreased protein synthesis in chronic alcoholism. (Mol Cell Biochem 115: 143-147, 1992)
Oxidative injury is believed to be a major factor in the pathogenesis of a variety of neurodegene... more Oxidative injury is believed to be a major factor in the pathogenesis of a variety of neurodegenerative diseases. Additionally, the mode of cell death in oxidant-stressed cells can vary. The present study was conducted to evaluate the use of a primary neuronal cell-based bioassay in which different modes of oxidant-induced cell death could be studied and in which putative neuroprotective agents could be screened. Addition of 50 AM H 2 O 2 to primary cortical neuronal cultures for 1 h under normal ATP conditions resulted in approximately 40% cell death, almost exclusively of an apoptotic nature. In this condition, cell death was effectively blocked by GM1 ganglioside, the semi-synthetic ganglioside derivative LIGA20, the dopamine receptor agonist pramipexole (PPX) and the caspase inhibitor Z-VAD-FMK but not by the poly (ADP-ribose) polymerase (PARP) inhibitor 3-aminobenzamide (3-AB). Pretreatment of cells with 0.01 AM oligomycin for 45 min prior to addition of 50 AM H 2 O 2 caused significant ATP depletion and approximately the same amount of cell death as H 2 O 2 alone. However, under these conditions, cell death was primarily non-apoptotic in nature and GM1, LIGA20 and Z-VAD-FMK had no protective effects. In contrast, AB and PPX effectively blocked cell death. These results suggest that cellular ATP plays a critical role in determining the mode of cell death in primary neurons and that these types of in vitro models may provide a useful system for screening putative neuroprotective agents.
The large cell doses (&am... more The large cell doses (>1 × 10(6) cells/kg) used in clinical trials with mesenchymal stem/stromal cells (MSC) will require an efficient production process. Moreover, monitoring and control of MSC ex-vivo expansion is critical to provide a safe and reliable cell product. Bioprocess engineering approaches, such as bioreactor technology, offer the adequate tools to develop and optimize a cost-effective culture system for the rapid expansion of human MSC for cellular therapy. Herein, a xenogeneic (xeno)-free microcarrier-based culture system was successfully established for bone marrow (BM) MSC and adipose tissue-derived stem/stromal cell (ASC) cultivation using a 1L-scale controlled stirred-tank bioreactor, allowing the production of (1.1 ± 0.1) × 10(8) and (4.5 ± 0.2) × 10(7) cells for BM MSC and ASC, respectively, after 7 days. Additionally, the effect of different percent air saturation values (%Airsat ) and feeding regime on the proliferation and metabolism of BM MSC was evaluated. No significant differences in cell growth and metabolic patterns were observed under 20% and 9%Airsat . Also, the three different feeding regimes studied-(i) 25% daily medium renewal, (ii) 25% medium renewal every 2 days, and (iii) fed-batch addition of concentrated nutrients and growth factors every 2 days-yielded similar cell numbers, and only slight metabolic differences were observed. Moreover, the immunophenotype (positive for CD73, CD90 and CD105 and negative for CD31, CD80 and HLA-DR) and multilineage differentiative potential of expanded cells were not affected upon bioreactor culture. These results demonstrated the feasibility of expanding human MSC from different sources in a clinically relevant expansion configuration in a controlled microcarrier-based stirred culture system under xeno-free conditions. The further optimization of this bioreactor culture system will represent a crucial step towards an efficient GMP-compliant clinical-scale MSC production system.
The immunomodulatory properties of mesenchymal stem cells (MSCs) make them attractive therapeutic... more The immunomodulatory properties of mesenchymal stem cells (MSCs) make them attractive therapeutic agents for a wide range of diseases. However, the highly demanding cell doses used in MSC clinical trials (up to millions of cells/kg patient) currently require labor intensive methods and incur high reagent costs. Moreover, the use of xenogenic (xeno) serum-containing media represents a risk of contamination and raises safety concerns. Bioreactor systems in combination with novel xeno-free medium formulations represent a viable alternative to reproducibly achieve a safe and reliable MSC doses relevant for cell therapy. The main goal of the present study was to develop a complete xeno-free microcarrier-based culture system for the efficient expansion of human MSC from two different sources, human bone marrow (BM), and adipose tissue. After 14 days of culture in spinner flasks, BM MSC reached a maximum cell density of (2.0 -0.2) • 10 5 cells$mL -1 (18 -1-fold increase), whereas adipose tissue-derived stem cells expanded to (1.4 -0.5) • 10 5 cells$mL -1 (14 -7-fold increase). After the expansion, MSC expressed the characteristic markers CD73, CD90, and CD105, whereas negative for CD80 and human leukocyte antigen (HLA)-DR. Expanded cells maintained the ability to differentiate robustly into osteoblast, adipocyte, and chondroblast lineages upon directed differentiation. These results demonstrated the feasibility of expanding human MSC in a scalable microcarrier-based stirred culture system under xeno-free conditions and represent an important step forward for the implementation of a Good Manufacturing Practicescompliant large-scale production system of MSC for cellular therapy.
DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme composed of a DNA-binding component ... more DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme composed of a DNA-binding component called Ku70/80 and a catalytic subunit called DNA-PKcs. Many investigators have utilized DNA-PKcs-deficient cells and cell lines derived from severe combined immunodeficiency (scid) mice to study DNA repair and apoptosis. However, little is known about the CNS of these mice. This study was carried out using primary neuronal cultures derived from the cerebral hemispheres of new-born wild-type and scid mice to investigate the effects of loss of DNA-PK function on neuronal maturation and survival. Purified neuronal cultures developed comparably in terms of neurite formation and expression of neuronal markers, but scid cultures showed a significant increase in the percentage of dying cells. Furthermore, when apoptosis was induced by staurosporine, scid neurons died more rapidly and in higher numbers. Apoptotic scid neurons exhibited nuclear condensation, DNA fragmentation and caspase-3 activation, but treatment with the general caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl) fluoromethyl ketone did not prevent staurosporine-induced apoptosis. We conclude that a DNA-PK deficiency in cultured scid neurons may cause an accumulation of DNA damage and increased susceptibility to caspase-independent forms of programmed cell death.
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Papers by Mohan Vemuri