Proceedings of the Annual Symposium on Regenerative Medicine(PASRM).

The primary characteristics of adult stem cells are maintaining prolonged quiescence, ability to self-renew and plasticity to differentiate into multiple cell types. These properties are evolutionarily conserved from fruit fly to humans. Similar to normal tissue repair in organs, the stem cell concept is inherently impregnated in the etiology of cancer. Tumors contain a minor population of tumor-initiating cells, called “cancer stem cells” that maintain some similarities in self-renewal and differentiation recognized as features of normal adult stem cells. Therefore, various methods developed originally for the analysis and characterization of adult stem cells are being extended to evaluate cancer stem cells. Relevant methods that are used generally across normal stem cells as well as cancer stem cells are summarized

The hypothesis that cancer could be a stem cell disease actually existed for several years before definitive ‘proof-of-concept’ experiments unequivocally established their involvement in initiating the tumorigenic state. Over the last decade, the combination of fluorescence-activated cell sorting with functional read-outs e.g. in vitro colony forming units in colony initiating assays, and competitive repopulating assays in animal models has enabled the prospective purification and enrichment of cancer stem cells (CSCs) in leukemia and also in certain solid tumors. Combination of two or more of these methods for validation of cancer stem cells appears to be a promising approach for the precise isolation and analysis of cancer stem cells. However, a major limitation for research in the field of stem cells and CSCs has been the lack of research resources in the form of in vitro models for these studies

Cancer is one of the leading causes of death among the human race. No valid modalities of treatment other than surgical treatment have been established for this disease. We aimed to identify and to characterize cancer using large number of human monoclonal antibodies (mAbs) which are specific against their surface for new molecular targeted immunotherapy. In order to find proper targets for therapeutic antibodies against cancers we developed a screening strategy. We used a huge phage library of human antibodies. At the first step we comprehensively isolated many monoclonal antibodies (mAbs) that specifically bound to surface of cancer cells. Development of ICOS (Isolation of antigen/antibody complexes through organic solvent) method allowed us to succeed in isolation of a huge number of mAbs with various characteristics (Y Akahori et al. 2009). At the next step we selected clones that showed tumor-specific staining patterns in immunohistochemical (IHC) analysis by using many fresh cancer tissues reseted. Many surgeons took part in this project. Finally the antigens recognized by these clones were identified by immunoprecipitation (IP) followed by analysis with mass (MS) spectrometry (G Kurosawa et al. 2009). We have succeeded in identification of 29 tumor-associated antigens (TAAs) and in isolation of 441 human mAbs that specifically bound to one of the 29 TAAs (G Kurosawa et al. 2008). In these screenings of the library, rounds of the selection process, mixing of cells and phage particles centrifugation growth of phages, were repeated three to four times in each screening. Therefore, numbers of phages of the clones whose antigens were abundantly present on the cell surface increased during the screenings. Recently we developed a new method for isolation of clones whose antigens were less abundantly present on the cell surface. Hence, we would like to talk on these methodology and discuss regarding this “A novel antibody based approach to Cancer Treatment” in this plenary session.

Background

There is increasing evidence that the transplanted bone marrow stromal cells (BMSC) significantly promote functional recovery after central nervous system (CNS) damage in the animal models of various kinds of CNS disorders, including cerebral infarct, brain contusion and spinal cord injury. However, there are several shortages of information when considering clinical application of BMSC transplantation for patients with neurological disorders. In this paper, therefore, we discuss what we should clarify to establish cell transplantation therapy in clinical situation and describe our recent works for this purpose.

Methods and Results

The BMSC have the ability to alter their gene expression profile and phenotype in response to the surrounding circumstances and to protect the neurons by producing some neurotrophic factors. They also promote neurite extension and rebuild the neural circuits in the injured CNS. Using optical imaging and MRI techniques, the transplanted BMSC can non-invasively be tracked in the living animals for at least 8 weeks after transplantation. Functional imaging such as PET scan may have the potential to assess the beneficial effects of BMSC transplantation. The BMSC can be expanded using the animal protein-free culture medium, which would maintain their potential of proliferation, migration, and neural differentiation.

Conclusion

It is urgent issues to develop clinical imaging technique to track the transplanted cells in the CNS and evaluate the therapeutic significance of BMSC transplantation in order to establish it as a definite therapeutic strategy in clinical situation in the future

BACKGROUND

Recent studies have shown that mesenchymal stem cells isolated from post natal human dental pulp, (Dental pulp stem cells-DPSCs) which is from permanent teeth and SHED (stem cells from human exfoliated deciduous teeth), the Periodontal ligament stem cells (PDLSC) and Stem cells from root Apical papilla(SCAP)have the potential to differentiate into cells of a variety of tissues including heart, muscle, cartilage, bone, nerve, salivary glands, teeth etc(. This multipotential ability of DPSCs is being researched for clinical application for treating a variety of diseases like myocardial infarction, muscular dystrophy, neurodegenerative disorders, cartilage replacement, tooth regeneration and for repair of bone defects to mention a few. Moreover, the isolation of stem cells from teeth is minimally invasive, readily accessible and the non immunogenic characteristic of dental stem cells has paved the way for efforts to store the exfoliated deciduous teeth or milk teeth which is usually discarded, for use in the future. In this study we have isolated and expanded in vitro, the cells obtained from human dental pulp.

METHODS AND RESULTS

After obtaining written informed consent, 24 teeth that were extracted for therapeutic or cosmetic reasons from 16 patients were used in this study. The specimens were transported from the clinic to NCRM lab taking 6 to 48 Hrs. For removal of the pulp tissue, the teeth were split obliquely at the Cementoenamel junction and the pulp tissue was isolated using brooches. The extracted pulp tissues were subjected to digestion using Collagenase type- I and type II at 37°C for 15- 30 minutes. The digested cells were filtered with 70μm filter and centrifuged at 1800 rpm for 10 minutes. The pellet was then suspended in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 supplemented with 15% fetal bovine serum , 100 U/ml penicillin, 100 μg/ml streptomycin,2 m M L -glutamine, and 2 m M nonessential amino acids ( .Cell counting was done by Trypan Blue dye exclusion method and the cells were seeded in 6 well culture plates. The plates with cells were incubated at 37°C with 5% CO2 for varying periods from 14 days-28 days. The cells were observed daily and media change was done every three days.

RESULTS

Viable Dental Pulp tissue-cells were obtained after transportation of up to 48 hrs and the in vitro growth of cells was initially slow but colonies were identified from the 10th day onwards. The cells were harvested at different intervals of 14-28 days for each sample based on their growth and subjected to H & E staining. The H & E staining of the cultured cells of all the samples showed positive results.

CONCLUSION

We are able to transport extracted teeth and derive viable dental pulp tissue cells after enzymatic digestion and multiply them in culture after a maximum of 48 hrs after transportation. The cells could be grown in culture with a morphology resembling dental pulp stem cells while in culture expansion and in H&E studies. Further characterization of the cells is necessary to confirm their Stemness.

The Enteric Nervous system (ENS) is a part of the Peripheral nervous system (PNS) that controls the peristaltic activity of the gut wall which is essential for propulsion of food in the digestive tract. It is composed of a large number of neurons and glial cells, distributed throughout the length of the gut. These ganglion cells develop from the neural crest in the embryo. Failure of complete colonization of the gut by these enteric neural crest cells during early development of life results in absence of ganglia or neurons in a portion of the gut, usually the colon which leads to aperistaltis and severe intestinal obstruction. This is known as Hirschsprung’s disease (HSCR) also known as congenital megacolon. HSCR affects 1 in 4500 newborns (. It appears either sporadically or has a familial basis and is often associated with other developmental defects. The main forms of treatment of HSCR are surgical resection of the aganglionic segment and pull through of the normal bowel. At present research is aimed at developing Cell based therapies for replacement of ganglion cells or enteric neuronal cells in the aganglionic portion of the gut thus aiming at restoring the function of the gut (. In this study we have isolated, in vitro expanded and characterized the Enteric Neuronal cells derived from human gut full thickness biopsy samples.

The postnatal gut full thickness biopsy samples of size 2-4 mm were obtained using from 13 patients undergoing gut resection surgery after informed consent. The samples were washed in Phosphate Buffer saline and using forceps, the outer smooth muscle layers along with the myenteric plexus were peeled off from the underlying tissue as strips. The strips were washed in Phosphate Buffer saline (PBS) and treated with 1mg/ml Collagenase/Dispase mixture in PBS for 30-45 min at 37°C. The digested cells were filtered with 70μm filter and the cell suspensions were centrifuged at 1800rpm for 10 mins. The pellet obtained was suspended in DMEM/F12 medium supplemented with penicillin (100U/ml), streptomycin (100 μg/ml), L-glutamine (2 mmol/L), growth factors like bFGF (20ng/ml) and EGF (20ng/ml)(2). Cell counting was done by Trypan Blue dye exclusion method and the cells were seeded in cell culture dishes coated with Fibronectin. The flasks with cells were incubated at 37°C with 5% CO2 for varying periods from 18 days-28 days. The cells were observed daily and media change was done every 2-3 days.

In all the samples, the Neurosphere like bodies (NLBs) were observed in the culture from 10th day onwards which were then subjected to histological and immunohistochemical studies. H&E staining showed positive for neural cells and Immunohistochemistry yielded positive for S-100, normally present in cells derived from the neural crest and Neuron Specific Enolase (NSE) a neuronal specific marker.

We could successfully isolate and expand Human Enteric Neuronal cells from postnatal gut biopsy samples. Further research is warranted to utilize these Enteric Neuronal Cells for Cell based therapies to treat Hirschsprung’s disease.

Recent studies have indicated that bone marrow stromal cells (BMSC) have the potential to improve neurological function when transplanted into animal model of central nervous system (CNS) disorders. However, there still exist several questions to solved prior to clinical application. In this study, therefore, we aimed to clarify the optimal delivery route of BMSC transplantation over a reasonable time window.

MATERIALS AND METHODS

The rats were subjected to permanent middle cerebral artery occlusion. The BMSC were labeled with quantum dot (QD) 800. The labeled BMSC were transplanted into the infarct brain directly or intravenously at 7 days after the insult. Motor function was serially assessed. The BMSC were also tracked using near infrared (NIR) fluorescence imaging technique every week. The fate of the transplanted BMSC was examined at 5 weeks after transplantation, using Immunohistochemistry.

Direct, but not intravenous, transplantation of BMSC significantly enhanced functional recovery. NIR fluorescence imaging could visualize their migration towards cerebral infarct in directly, but not intravenously, injected animals. The findings were supported on histological analysis. Thus, the BMSC were widely engrafted in the infarct brain in the directly injected animals, but few BMSC were observed in the intravenously injected ones.

This study strongly suggests that direct transplantation of BMSC may be more beneficial in treating patients with ischemic stroke than their intravenous transplantation. Therapeutic time window must be called into account when considering the route of BMSC transplantation.

Vascular endothelial cells play a major role in wound healing and also in growth of the tumors. Angiogenesis can be a target for treating diseases that are due to either poor vascularisation or decreased blood supply as in stroke, ulcers, heart disease, etc or abnormal and increased vasculature like in tumours. Application of specific compounds that may inhibit or induce the creation of new blood vessels in the body may help in the treatment of such diseases (. Ex vivo generation of blood vessels may offer an excellent alternative to the synthetic valves that are being currently used in cardiology. Micro gravity also referred to, as weightlessness is not essentially zero gravity but rather minimal gravity. According to cell type, micro gravity causes variety of changes in proliferation and differentiation of cells while also affecting the migration of cells and cellular functions (. Siamwala et al from AUKBC have already studied the effects of microgravity on the microvascular endothelial cells from bovine lung and macrovascular endothelial cells from the bovine pulmonary artery. It was observed that the proliferation and migration of macrovascular endothelial cells were increased in microgravity (. Nitric oxide production was also studied and observed that microgravity treatment did not change nitric oxide production by microvascular endothelial cells (.

OBJECTIVES

Isolation and Comparison of culture characteristics of Human microvascular endothelium cultured conventionally and in novel nanomaterial scaffold and further study the morphological and molecular characteristics of microvascular endothelial cells under normal gravity against simulated micro gravity.

The human Omentum samples were obtained using surgical procedures after informed consent. The microvascular endothelial cells were isolated following the protocol described by Scott et al (. The isolated cells were seeded in two groups; Group I - Cells in Nano Polymer, Overlaid M199 medium +20%FBS and, Group II - Cells in Conventional M199 medium+20%FBS. The cultures were maintained at 37C with 5%CO2. The medium was changed every 3 days thereafter. The cells were observed regularly for their morphological characteristics and images were documented. At day 7 and day 13 the cells were harvested and the cells were subjected to two assays to confirm the presence of nitric oxide (NO) in samples, which will in turn confirm the presence of endothelial cells. In the first assay the isolated cell suspension was probed with diaminorhodamine-4M (DAR-4FM). In the second assay, the total nitrite level in the isolated cells suspended media was measured using Griess Assay protocol.

A 21% increase in number of NO positive cells was observed in the cells cultured on the novel nanopolymer, while a 1.7 fold increase in nitrite production was detected in Group I in comparison to that of Group II.

Our data suggests the presence of more NO driven nitrite in the Microvascular endothelial cells cultured in the nanopolymer. In the next phase of the study, the cells thus isolated from both the groups will be subjected to simulated microgravity of 10-3 g in a clinostat at the AUKBC Vascular Biology Lab and the morphological and molecular characteristics of Microvascular endothelial cells will be studied to identify whether the effects of microgravity on Microvascular endothelial cells is organ specific.

Patients with complete spinal cord injury at cervical level, mostly lead a life with quadriplegia. We report a case of 22 year old male, who became quadriplegic after C4-C5 injury and has improved after five injections of in-vitro expanded autologous bone marrow mononuclear cells (ABMMNC).

The patient developed complete quadriplegia following fall from height. MRI suggested C5 burst fracture with retropulsion of C5 vertebrae and cord contusion at C4-C5 and underwent decompression and fixation four days post injury. After surgical fixation, bone marrow was harvested twice and a portion of the MNCs were expanded in-vitro and injected and another portion was cryopreserved which were later expanded and injected through lumbar puncture. The first injection was given 13 days after surgery. The first specimen of BM had CD34 of 0.18% and was injected twice; once immediately after harvesting (CD34:1.57%) and later after cryopreservation for 11 months (CD34: 3.33%). The second specimen had a CD34 of 0.52% and was injected thrice; twice after harvesting with CD34 of 1.28% and 6.23% respectively and later after cryopreservation for four months (CD34: 14.03%). The expansion was done in a GMP compliant clean room using autologous serum and the specimens were subjected to aerobic culture and Endotoxin tests before each administration.

Investigations done (i)Post injury, (ii) Post decompression and (iii) one year (iv) One and half year after the injury have shown objective improvements with Light touch score (20-22-40-48), Pin prick score (20-20-32-40), Anal sensations (Nil-Nil-Present), Motor Score (11-11-22-26), Sensory level (C6-C6-C6-C6), Motor level (C5-C5-C6-C6), Neurological level (C5-C6-C6-C6), Spinal cord independence measure (9-12-45-50) and Barthel index (0-0-30-40). Repeat MRI show gliosis at C5. The ASIA score has improved from A to B with no adverse reactions.

Multiple intrathecal injections of in-vitro expanded ABMMNCs in cervical-spinal cord injury was found to be safe and gradual objective improvements have been observed over a year and a half without any adverse outcome.

Following traumatic vertebral injuries and resultant spinal cord injury, most patients are doomed to a life either of quadriplegia or paraplegia. Current treatment option is limited to the stabilization of the vertebral fracture along with medications to prevent secondary damage leading to further deterioration and wishful waiting for recovery. In most instances recovery is insignificant. Safety of intrathecal injection of autologous bone marrow stem cells is proven but its efficacy varies between patients (. Intralesional application has been reported to be more efficacious than intrathecal application (. We have analyzed our experience in human patients followed up for 3 year period and have found several grey areas in spinal cord injury( one of them is to explore the differences between Intrathecal and intralesional application of stem cells with and without scaffolds in the latter technique. Towards achieving this goal we started a pilot study in animals where instead of post-vertebral fixation intrathecal injection, we have performed intralesional application of autologous BMSC along with scaffolds (. These scaffolds not only help retain the transplanted cells at the site of injury but also allow more neural precursors to grow compared to application without scaffolds (. This study analyses the data retrospectively to plan further prospective studies with a view to improvise the results.

Study 1

100 to 120 ml of Bone marrow was tapped from the right posterior iliac crest under local anesthesia from human spinal injury victims (n=108; 76 males, 32 females) about 3 weeks to 18 months after surgical fixation of the vertebrae. The Level of injury was varied- Cervical (13 patients.) Upper Thorax- T1-T7 (35 patients) Lower thorax T8-T12 (46 patients) Lumbar (2 patients.) Age Group Range: 8 yrs to 55 yrs. The bone marrow mononuclear cells were processed under cGMP SOP’s Class 10000 clean room and class 100 Biosafety hood as reported earlier ( and were injected intrathecally into the subarachnoid space of the patients below L2 level after endotoxin tests and confirming CD34 status using flow cytometry.

Study 2

20-30ml of Bone marrow was tapped from the right posterior iliac crest under local anesthesia of canine spinal injury victims immediately after the injury and the bone marrow processed as reported earlier ( were injected intralesionally embedded in thermoreversible hydrogel scaffolds at the site of the injury after endotoxin tests and CD34 analysis using flow cytometry. Both the animals had an Olby score of 1 with no CP reflex, Patellar reflex and deep pain reflex.

Study-2

The first canine died due to cystitis on the 7th day after the procedure and the cause of death was confirmed to be unrelated to the procedure by autopsy that revealed co-morbid conditions like cystitis, nephritis and transmissible venereal tumor. Histopathology of the engrafted area revealed sustainability of aggregated stem cells that were transplanted revealing an ideal biocompatibility of the construct prepared with bone marrow mononuclear cells and polymer hydrogel for spinal cord regeneration in dogs. The second canine had complete recovery of hind limb function and coordination after 6 months, Olby score has improved to 14 (normal 15) with normal bowel and bladder control.

DISCUSSION

It was hypothesized that intralesional application at time of surgery may be superior to Intrathecal application of bone marrow stem cells simply because the former technique ensured the delivery of a higher proportion of cells in the damaged area. It is possible that the cells injected intrathecally are carried along with the CSF to parts other than that damaged as well. This has been supported by study 2 when it was applied with scaffold. The injury model used in study 2 is a natural traumatic model and is more akin to real life than any other controlled spinal cord model that one could create in the lab. This study 2 which is still in process allows for the animal to live to its entire course enabling us to follow up the neurological recovery of the patient and on its death perform an autopsy to not only determine the cause of death but to also examine the fate of stem cells injected intralesionally. We hope to determine the percentage of stem cells remaining as stem cells and to determine the nature and magnitude of histopathological changes that might have taken place which facilitated /non facilitated the recovery in the study animals

It is understood from the study 1, that the factors determining outcome are multiple and includes the age of patients, level of injury, time interval between injury and ABMMC injection, dosage of stem cells injected and all these need to be evaluated in future studies. More studies are necessary to ascertain the efficacy. Safety of both intrathecal and intralesional injection with scaffold have been proven in this studies. Inclusion of larger number of cases with a long term follow up is necessary to know the efficacy of intralesional therapy with scaffolds.

Parameters	4-6 months follow-up(1)	12-36 months follow-up(8)
Follow up attrition rate:	14.02%	69%
Motor power improvement (atleast 2 grade of power post injection)	14.11%	7.69%
Motor power improvement resulting in functional recovery	4.70% 3 walk with support, 1 walking without support	5.13%
Subjective sensory improvement	16.97%	25.64%
Abnormal sensation	0%	0%
Autonomic improvement by Urodynamic studies	9.41%	10.26%