Robust platform for inline Raman monitoring and control of perfusion cell culture.

Perfusion cell culture has been gaining increasing popularity for biologics manufacturing due to benefits such as smaller footprint, increased productivity, consistent product quality and manufacturing flexibility, cost savings, and so forth. Process Analytics Technologies tools are highly desirable for effective monitoring and control of long-running perfusion processes. Raman has been widely investigated for monitoring and control of traditional fed batch cell culture process. However, implementation of Raman for perfusion cell culture has been very limited mainly due to challenges with high-cell density and long running times during perfusion which cause extremely high fluorescence interference to Raman spectra and consequently it is exceedingly difficult to develop robust chemometrics models. In this work, a platform based on Raman measurement of permeate has been proposed for effective analysis of perfusion process. It has been demonstrated that this platform can effectively circumvent the fluorescence interference issue while providing rich and timely information about perfusion dynamics to enable efficient process monitoring and robust bioreactor feed control. With the highly consistent spectral data from cell-free sample matrix, development of chemometrics models can be greatly facilitated. Based on this platform, Raman models have been developed for good measurement of several analytes including glucose, lactate, glutamine, glutamate, and permeate titer. Performance of Raman models developed this way has been systematically evaluated and the models have shown good robustness against changes in perfusion scale and variations in permeate flowrate; thus models developed from small lab scale can be directly transferred for implementation in much larger scale of perfusion. With demonstrated robustness, this platform provides a reliable approach for automated glucose feed control in perfusion bioreactors. Glucose model developed from small lab scale has been successfully implemented for automated continuous glucose feed control of perfusion cell culture at much larger scale.

Automated Online-Sampling Multidimensional Liquid Chromatography with Feedback-Control Capability as a Framework for Real-Time Monitoring of mAb Critical Quality Attributes in Multiple Bioreactors.

Real-time monitoring of biopharmaceutical reactors is becoming increasingly important as the processes become more complex. During the continuous manufacturing of monoclonal antibodies (mAbs), the desired mAb product is continually created and collected over a 30 day process, where there can be changes in quality over that time. Liquid chromatography (LC) is the workhorse instrumentation capable of measuring mAb concentration as well as quality attributes such as aggregation, charge variants, oxidation, etc. However, traditional offline sampling is too infrequent to fully characterize bioprocesses, and the typical time from sample generation to data analysis and reporting can take weeks. To circumvent these limitations, an automated online sampling multidimensional workflow was developed to enable streamlined measurements of mAb concentration, aggregation, and charge variants. This analytical framework also facilitates automated data export for real-time analysis of up to six bioreactors, including feedback-controlling capability using readily available LC technology. This workflow increases the data points per bioreactor, improving the understanding of each experiment while also reducing the data turnaround time from weeks to hours. Examples of effective real-time analyses of mAb critical quality attributes are illustrated, showing substantial throughput improvements and accurate results while minimizing labor and manual intervention.

Nkx6.1 enhances neural stem cell activation and attenuates glial scar formation and neuroinflammation in the adult injured spinal cord.

Nkx6.1 plays an essential role during the embryonic development of the spinal cord. However, its role in the adult and injured spinal cord is not well understood. Here we show that lentivirus-mediated Nkx6.1 expression in the adult injured mouse spinal cord promotes cell proliferation and activation of endogenous neural stem/progenitor cells (NSPCs) at the acute phase of injury. In the chronic phase, Nkx6.1 increases the number of interneurons, reduces the number of reactive astrocytes, minimizes glial scar formation, and represses neuroinflammation. Transcriptomic analysis reveals that Nkx6.1 upregulates the sequential expression of genes involved in cell proliferation, neural differentiation, and Notch signaling pathway, downregulates genes and pathways involved in neuroinflammation, reactive astrocyte activation, and glial scar formation. Together, our findings support the potential role of Nkx6.1 in neural regeneration in the adult injured spinal cord.

Gsx1 promotes locomotor functional recovery after spinal cord injury.

Promoting residential cells, particularly endogenous neural stem and progenitor cells (NSPCs), for tissue regeneration represents a potential strategy for the treatment of spinal cord injury (SCI). However, adult NSPCs differentiate mainly into glial cells and contribute to glial scar formation at the site of injury. Gsx1 is known to regulate the generation of excitatory and inhibitory interneurons during embryonic development of the spinal cord. In this study, we show that lentivirus-mediated expression of Gsx1 increases the number of NSPCs in a mouse model of lateral hemisection SCI during the acute stage. Subsequently, Gsx1 expression increases the generation of glutamatergic and cholinergic interneurons and decreases the generation of GABAergic interneurons in the chronic stage of SCI. Importantly, Gsx1 reduces reactive astrogliosis and glial scar formation, promotes serotonin (5-HT) neuronal activity, and improves the locomotor function of the injured mice. Moreover, RNA sequencing (RNA-seq) analysis reveals that Gsx1-induced transcriptome regulation correlates with NSPC signaling, NSPC activation, neuronal differentiation, and inhibition of astrogliosis and scar formation. Collectively, our study provides molecular insights for Gsx1-mediated functional recovery and identifies the potential of Gsx1 gene therapy for injuries in the spinal cord and possibly other parts of the central nervous system.

A novel mouse model for the study of endogenous neural stem and progenitor cells after traumatic brain injury.

Traumatic brain injury (TBI) is a leading cause of death and disability in the US. Neural stem/progenitor cells (NSPCs) persist in the adult brain and represent a potential cell source for tissue regeneration and wound healing after injury. The Notch signaling pathway is critical for embryonic development and adult brain injury response. However, the specific role of Notch signaling in the injured brain is not well characterized. Our previous study has established a Notch1CR2-GFP reporter mouse line in which the Notch1CR2 enhancer directs GFP expression in NSPCs and their progeny. In this study, we performed closed head injury (CHI) in the Notch1CR2-GFP mice to study the response of injury-activated NSPCs. We show that CHI induces neuroinflammation, cell death, and the expression of typical TBI markers (e.g., ApoE, Il1b, and Tau), validating the animal model. In addition, CHI induces cell proliferation in GFP+ cells expressing NSPC markers, e.g., Notch1 and Nestin. A significant higher percentage of GFP+ astrocytes and GABAergic neurons was observed in the injured brain, with no significant change in oligodendrocyte lineage between the CHI and sham animal groups. Since injury is known to activate astrogliosis, our results suggest that injury-induced GFP+ NSPCs preferentially differentiate into GABAergic neurons. Our study establishes that Notch1CR2-GFP transgenic mouse is a useful tool for the study of NSPC behavior in vivo after TBI. Unveiling the potential of NSPCs response to TBI (e.g., proliferation and differentiation) will identify new therapeutic strategy for the treatment of brain trauma.

A biodegradable hybrid inorganic nanoscaffold for advanced stem cell therapy.

Stem cell transplantation, as a promising treatment for central nervous system (CNS) diseases, has been hampered by crucial issues such as a low cell survival rate, incomplete differentiation, and limited neurite outgrowth in vivo. Addressing these hurdles, scientists have designed bioscaffolds that mimic the natural tissue microenvironment to deliver physical and soluble cues. However, several significant obstacles including burst release of drugs, insufficient cellular adhesion support, and slow scaffold degradation rate remain to be overcome before the full potential of bioscaffold-based stem-cell therapies can be realized. To this end, we developed a biodegradable nanoscaffold-based method for enhanced stem cell transplantation, differentiation, and drug delivery. These findings collectively support the therapeutic potential of our biodegradable hybrid inorganic (BHI) nanoscaffolds for advanced stem cell transplantation and neural tissue engineering.

The growing role of precision and personalized medicine for cancer treatment.

Cancer is a devastating disease that takes the lives of hundreds of thousands of people every year. Due to disease heterogeneity, standard treatments, such as chemotherapy or radiation, are effective in only a subset of the patient population. Tumors can have different underlying genetic causes and may express different proteins in one patient versus another. This inherent variability of cancer lends itself to the growing field of precision and personalized medicine (PPM). There are many ongoing efforts to acquire PPM data in order to characterize molecular differences between tumors. Some PPM products are already available to link these differences to an effective drug. It is clear that PPM cancer treatments can result in immense patient benefits, and companies and regulatory agencies have begun to recognize this. However, broader changes to the healthcare and insurance systems must be addressed if PPM is to become part of standard cancer care.

Differential Gene Regulation and Tumor-Inhibitory Activities of Alpha-, Delta-, and Gamma-Tocopherols in Estrogen-Mediated Mammary Carcinogenesis.

Despite experimental evidence elucidating the antitumor activities of tocopherols, clinical trials with α-tocopherol (α-T) have failed to demonstrate its beneficial effects in cancer prevention. This study compared the chemopreventive efficacy of individual tocopherols (α-, δ-, and γ-T) and a γ-T-rich tocopherol mixture (γ-TmT) in the August-Copenhagen Irish (ACI) rat model of estrogen-mediated mammary cancer. Female ACI rats receiving 17ß-estradiol (E2) implants were administered with 0.2% α-T, δ-T, γ-T, or γ-TmT for 30 weeks. Although α-T had no significant effects on mammary tumor growth in ACI rats, δ-T, γ-T, and γ-TmT reduced mammary tumor volume by 51% (P < 0.05), 60% (P < 0.01), and 59% (P < 0.01), respectively. Immunohistochemical analysis revealed that δ-T, γ-T, and γ-TmT reduced levels of the cell proliferation marker, proliferating cell nuclear antigen, in the rat mammary tumors. To gain further insight into the biological functions of different forms of tocopherols, RNA-seq analysis of the tumors was performed. Treatment with γ-T induced robust gene expression changes in the mammary tumors of ACI rats. Ingenuity Pathway Analysis identified "Cancer" as a top disease pathway and "Tumor growth" and "Metastasis" as the top signaling pathways modulated by γ-T. Although the results need further functional validation, this study presents an unbiased attempt to understand the differences between biological activities of individual forms of tocopherols at the whole transcriptome level. In conclusion, δ-T and γ-T have superior cancer preventive properties compared to α-T in the prevention of estrogen-mediated mammary carcinogenesis. Cancer Prev Res; 10(12); 694-703. ©2017 AACR.

Decrypting the PAK4 transcriptome profile in mammary tumor forming cells using Next Generation Sequencing.

The p-21 Activated Kinase 4 (PAK4) protein kinase is implicated in many cancers, including breast cancer. Overexpression of PAK4 is sufficient to cause mouse mammary epithelial cells (iMMECs) to become tumorigenic. To gain insight into the long-term gene expression changes that occur downstream to PAK4, we performed Next Generation Sequencing of RNA collected from PAK4 overexpressing iMMECs and wild-type iMMECs. We identified a list of genes whose expression levels were altered in response to PAK4 overexpression in iMMECs. Some of these genes, including FoxC2 and ParvB, are consistent with a role for PAK4 in cancer. In addition, PAK4 regulates many genes that are frequently associated with the inflammatory response, raising the possibility that there is a connection between PAK4, inflammation, and the tumor microenvironment. This study delineates the PAK4 transcriptome profile in transformed mammary cells and can provide translational utility in other types of cancers as well.