Engineering bio-mimetic humanized neurological constructs using acellularized scaffolds of cryopreserved meningeal tissues.

Spinal cord injury (SCI) is one of the most precarious conditions which have been one of the major reasons for continuous increasing mortality rate of SCI patients. Currently, there is no effective treatment modality for SCI patients posing major threat to the scientific and medical community. The available strategies don't mimic with the natural processes of nervous tissues repair/regeneration and majority of the approaches may induce the additional fibrotic or immunological response at the injury site and are not readily available on demand. To overcome these hurdles, we have developed a ready to use bioengineered human functional neurological construct (BHNC) for regenerative applications in SCI defects. We used cryopreserved meningeal tissues (CMT) for bioengineering these neurological constructs using acellularization and repopulation technology. The technology adopted herein generates intact neurological scaffolds from CMT and retains several crucial structural, biochemical and mechanical cues to enhance the regenerative mechanisms. The neurogenic differentiation on CMT scaffolds was almost similar to the freshly prepared meningeal scaffolds and mimics with the natural nervous tissue developmental mechanisms which offer intact 3D-microarchitecture and hospitable microenvironment enriched with several crucial neurotrophins for long-term cell survival and function. Functional assessment of developed BHNC showed highly increased positive staining for pre-synaptic granules of Synapsis-1 along with MAP-2 antibody with punctuate distribution in axonal regions of the neuronal cells which was well supported by the gene expression analysis of functional transcripts. Given the significant improvement in the field may enable to generate more such ready to use functional BHNC for wider applicability in SCI repair/regeneration.

Intraperitoneal transplantation of bioengineered humanized liver grafts supports failing liver in acute condition.

Acute liver failure (ALF) is one of the most devastating fatal conditions which have posed crucial challenges to the clinicians and researchers for identifying permanent cure. Currently liver transplantation has been considered as the only managerial option. However it's wider applicability has been limited owing to non-availability of quality donor organs, cost-intensiveness, surgical hitches, life-long use of immunosuppressive drugs and long-term complications. Since last decades, several liver support systems have been developed for the management of failing liver in acute condition. However, the major limitation has been the lack of natural biological support and long-term survival of the grafts post-transplantation. Repopulation of decellularized xenogeneic organs is one of the emerging technologies for development of humanized neo-organs for demanding regenerative application. However, the earlier reported studies do not fulfil the insistence to provide immunologically tolerable humanized liver grafts for clinical applications. Here we demonstrate an efficient approach to generate transplantable humanized liver grafts which provides long-term support to the failing liver in Acute Liver Failure (ALF) animal models. These bioengineered humanized liver tissue grafts expresses several liver specific transcripts and performed crucial synthetic (albumin production) and detoxification (urea synthesis) functions at comparative level to normal liver. Intraperitoneal transplantation of these humanized liver grafts offered favourable microenvironment to exchange toxic substances across the barrier during ALF condition and provided long-term survival and function of the graft. In summary, the results of present study provide a first proof of concept in pre-clinical ALF animal model for the applicability of these bioengineered humanized livers in the management of failing liver on demand and may be considered as potential bridge to liver transplantation.

Bioengineered functional humanized livers: An emerging supportive modality to bridge the gap of organ transplantation for management of end-stage liver diseases.

End stage liver diseases (ESLD) represent a major, neglected global public health crisis which requires an urgent action towards finding a proper cure. Orthotropic liver transplantation has been the only definitive treatment modality for ESLD. However, shortage of donor organs, timely unavailability, post-surgery related complications and financial burden on the patients limits the number of patients receiving the transplants. Since last two decades cell-based therapies have revolutionized the field of organ/tissue regeneration. However providing an alternative organ source to address the donor liver shortage still poses potential challenges. The developments made in this direction provide useful futuristic approaches, which could be translated into pre-clinical and clinical settings targeting appropriate applications in specific disease conditions. Earlier studies have demonstrated the applicability of this particular approach to generate functional organ in rodent system by connecting them with portal and hepatic circulatory networks. However, such strategy requires very high level of surgical expertise and also poses the technical and financial questions towards its future applicability. Hence, alternative sites for generating secondary organs are being tested in several types of disease conditions. Among different sites, omentum has been proved to be more appropriate site for implanting several kinds of functional tissue constructs without eliciting much immunological response. Hence, omentum may be considered as better site for transplanting humanized bioengineered ex vivo generated livers, thereby creating a secondary organ at intra-omental site. However, the expertise for generating such bioengineered organs are limited and only very few centres are involved for investigating the potential use of such implants in clinical practice due to gap between the clinical transplant surgeons and basic scientists working on the concept evolution. Herein we discuss the recent advances and challenges to create functional secondary organs through intra-omental transplantation of ex vivo generated bioengineered humanized livers and their further application in the management of ESLD as a supportive bridge for organ transplantation.

Bioengineering Human Neurological Constructs Using Decellularized Meningeal Scaffolds for Application in Spinal Cord Injury.

Spinal cord injury (SCI) is one of the most devastating conditions echoes with inflammation, enhanced fibrosis and larger axonal gaps due to destruction of neurological cells which has caused continuous increasing mortality rate of SCI patients due to absence of suitable treatment modalities. The restoration of structural and functional aspect of damaged neurological tissues at the lesion site in spinal cord has been challenging. Recent developments have showed tremendous potential of neural stem cell-based strategies to form a neuronal relay circuit across the injury gap which facilitates some levels of improvement in SCI condition. However, to provide better therapeutic responses, critical mass of grafted cells must survive for long-term and differentiate into neuronal cells with well-developed axonal networks. Hence, development of tissue specific biological neuronal constructs is highly desirable to provide mechanical and biological support for long-term survival and function of neurological cells within natural biological niche. In this study, we report development of a tissue specific neuronal constructs by culturing human neural precursor cells on decellularized meningeal scaffolds to provide suitable biological neuronal construct which can be used to support mechanical, structural and functional aspect of damaged spinal cord tissues. This particular tissue specific biological construct is immunologically tolerable and provides precisely orchestral three-dimensional platform to choreograph the long-distance axonal guidance and more organized neuronal cell growth. It passes sufficient mechanical and biological properties enriched with several crucial neurotrophins required for long-term survival and function of neurological cells which is required to form proper axonal bridge to regenerate the damaged axonal connectomes at lesion-site in SCI.

Molecular dynamics of pancreatic transcription factors in bioengineered humanized insulin producing neoorgan.

BACKGROUND: The present study has been aimed to identify molecular dynamics of pancreatic transcription factors (pTFs) during events of directed trans-differentiation of human hepatic progenitor cells (hHPCs) into insulin producing cells (InPCs) within bioengineered humanized neoorgan. The study demonstrates applicability of acellularized whole splenic scaffold (ASOS) to generate insulin producing humanized transplantable neoorgan through activation of pancreatic transcription factors. METHODS: An efficient acellularization process was developed for xenogeneic rat spleen using change in different gradients of reagents perfusion through splenic artery for varying time points. The acellularized xenogeneic spleen scaffold was characterized thoroughly for preservation of extra-cellular matrix and retention of organ specific vasculature and mechanical properties. Further scaffolds were sterilized and repopulated with hHPCs which were triggered using a stage wise induction with growth factors and hyperglycemic challenge for trans-differentiation into InPCs. Dynamics of pTFs alone or simultaneously during induction process was identified using gene expression analysis and immunological staining. RESULTS: The cells within the engineered neoorgan respond to growth factors and extrinsic hyperglycemic challenge and generate large number of InPCs under controlled dynamic regulation of pTFs. Highly controlled regulation of pTFs generates higher percentage of Nkx-6.1+/C-peptide+ cells within the engineered splenic scaffolds. Generation of high percentage of insulin and C-peptide positive cells in three-dimensional organ architecture responded better to hyperglycemic stimuli and produced higher quantity of insulin than 2D-culture system. CONCLUSION: The present study provides a novel platform for designing effective regenerative strategies using whole organ scaffolds to control hyperglycemia under tight regulation of pTFs using humanized neoorgan system.

Role of drug transporters and heat shock proteins during ethanol exposure to human neural precursor cells and its lineages.

INTRODUCTION: Ethanol exposure to developing brain may alter the growth and differentiation of neurological cells resulting in unfavorable pathologies. Earlier studies have provided very limited mechanistic insights of cellular and molecular mechanisms which do not mimic with human situation due to varying cell types and poses potential challenges for investigation. Therefore, the present study was undertaken to evaluate the role of ABC transporters and heat shock proteins mediated response in human neural precursor cells (NPCs) and its lineages during proliferation and lineage differentiation against ethanol exposure. METHODS: Effect of ethanol exposure was examined for neuronal cell survival and variation in cellular phenotype during neurospheres development and lineage differentiation. Generation of reactive oxygen species, and variation in cell cycle was identified along with transcriptional profiling for pluripotent markers (Nestin, NCAM, Sox-2, and Notch-2), drug transporters (ABCB1 and ABCG2) and stress protein (HSP70) during ethanol exposure. RESULTS: ABC transporters as well as HSP70 mRNA expression was higher during proliferation as compared to differentiation with chronic ethanol (1 M) exposure (p < 0.01). Ethanol exposure resulted in higher variability in size and shape of developing neurospheres and decreased ability to form new neurosphere colonies. Significant changes were observed in dendrite development due to late ethanol exposure (p < 0.0001). CONCLUSION: The present study demonstrated significant role of ABC transporters and HSP70 proteins in providing defense against ethanol-induced damage in human neurological cells. However, the over-expression of ABC transporter and HSP-70 proteins during such pathological conditions do not provide complete defense and additional strategies are required to repair the damage.

Enhanced neuroprotective effect of mild-hypothermia with VPA against ethanol-mediated neuronal injury.

INTRODUCTION: Progress in understanding pathophysiological mechanisms and the development of targeted regenerative strategies have been hampered by the lack of predictive disease models, specifically for the conditions to which affected cell types are inaccessible. The present study has aimed to unearth the role of valproic acid (VPA) and mild hypothermia (MH) as promising strategy to enhance the neuroprotective mechanisms in undifferentiated and differentiated human neural precursor cells (hNPCs) against ethanol-induced damage. METHODS: 5mM VPA alone or in combination with MH (33°C) was used to prevent the damage in proliferating and differentiating hNPCs. CD133+ve enriched hNPCs were cultured in vitro and exposed to 1M chronic ethanol concentration for 72h and followed by VPA and MH treatment for 24h. Morphometric analysis was performed to identify changes in neurospheres development and neuronal cell phenotypes. Flow cytometry and RT-qPCR analysis was performed to investigate alterations in key molecular pathways involved in cell survival and signaling. RESULTS: Combination of VPA with MH displayed higher proportion of neuronal cell viability as compared to single treatment. Combination treatment was most effective in reducing apoptosis and reactive oxygen species levels in both the undifferentiated and differentiated hNPCs. VPA with MH significantly improved neuronal cell phenotype, active chromatin modeling, chaperon and multi-drug resistant pumps activity and expression of neuronal signaling molecules. CONCLUSION: The study provided an efficient and disease specific in vitro model and demonstrated that combined treatment with VPA and MH activates several neuroprotective mechanisms and provides enhanced protection against ethanol-induced damage in cultured undifferentiated and differentiated hNPCs.

Protective Role of Hypothermia Against Heat Stress in Differentiated and Undifferentiated Human Neural Precursor Cells: A Differential Approach for the Treatment of Traumatic Brain Injury.

INTRODUCTION: The present study aimed to explore protective mechanisms of hypothermia against mild cold and heat stress on highly proliferative homogeneous human Neural Precursor Cells (NPCs) derived from Subventricular Zone (SVZ) of human fetal brain. METHODS: CD133+ve enriched undifferentiated and differentiated human NPCs were exposed to heat stress at 42°C. Then, Western-blot quantification was performed using Hsp-70 (70 kilodalton heat shock proteins) recombinant protein. Finally, changes in pluripotency and Hsp-70 expression were measured using immunofluorescence staining and RT-qPCR (Quantitative reverse transcription PCR) analysis, respectively. RESULTS: Heat stress resulted in abnormal neurospheres development. The apoptosis rate was enhanced during long-term in vitro culture of neurospheres. Neurogenic differentiation reduced and showed aberrent phenotypes during heat stress. After hypothermia treatment significant improvement in neurospheres and neuronal cell morphology was observed. CONCLUSION: Mild-hypothermia treatment induces attenuated heat shock response against heat stress resulting in induced HSP-70 expression that significantly improves structure and function of both undifferentiated human NPCs and differentiated neurons.

Pluripotent stem cells - a review of the current status in neural regeneration.

Pharmacological or neurosurgical therapies currently in practice to treat the damage in various neurodegenerative disorders are not efficient in preventing progression or cure of these progressive neurodegenerative processes. Recently, a new approach, cell therapy using stem cell, is being evaluated. However, the use of this therapy in the treatment of these neurological diseases is highly restricted, mainly owing to several technical difficulties and limitations. The strategy of isolation and characterization of neural stem cells from various sources will probably provide a major impetus and open up an interesting, novel therapeutic modality for several neurodegenerative disorders. The high regenerative potential of damaged neural tissues suggests that various embryonic/adult sources serve as a proxy for neural stem cells for cell-based therapy.

Neural stem cells & supporting cells--the new therapeutic tools for the treatment of spinal cord injury.

Stem cells play important role in the development and in the maintenance of specific tissues. They have been identified in majority of the organs like liver, blood, skin and intestine. Role of stem cells in regenerative medicine have been implicated in many chronic diseases. Stem cell research is a new opportunity to those patients whose organs are damaged or diseased. The discovery of stem cells in central and peripheral nervous system is relatively recent. Spinal cord injury is one of the major neurological disaster affecting mostly young lives. Stem cell transplantation in spinal cord injury patients have shown encouraging results. Different sources of stem cells are being exploited for spinal cord injury as well as other neurological disorders.