Rat bone marrow stromal cell transplantation ameliorates complete spinal cord injury induced sensorimotor dysfunctions and associated neurotransmitters

Traumatic spinal cord injury (SCI) leads to sensorimotor dysfunction with significant impact on the patient and their family’s quality of life, social, and economic status. There is no complete restorative treatment so far. Bone marrow stromal cells (BMSCs) have anti-inflammatory and neuroprotective effects and recently emerged as a therapeutic candidate for SCI repair. Here, we examined the role of rat BMSCs transplantation on thoracic (T11) complete SCI induced dysfunctions, namely hyperalgesia, allodynia, locomotion, spinal reflexes, and spinal neurotransmitters in rats. Pre-labelled BMSCs were injected on day 9 after SCI locally. We observed that BMSCs transplantation facilitate locomotor recovery (week 2-8) and attenuated hyperalgesia and allodynia to varying sensory stimuli (week 6-8) after SCI. In addition, spinal reflexes and neurotransmitters were affected significantly by complete SCI, which were partially restored by BMSCs transplantation. Histological analyses also revealed the presence of BMSCs at the injury site and appear to fill the lesion cavities, thereby significantly reducing the lesion volume. Our data shows the beneficial effects of BMSCs transplantation on complete SCI-induced sensorimotor functional deficits in rats.

Hair & skin derived progenitor cells: In search of a candidate cell for regenerative medicine.

Background & objectives: Skin is an established tissue source for cell based therapy. The hair follicle has been introduced later as a tissue source for cell based therapy. The ease of tissue harvest and multipotent nature of the resident stem cells in skin and hair follicle has promoted basic and clinical research in this area. This study was conducted to evaluate skin stem cells (SSCs) and hair follicle stem cells (HFSCs) as candidate cells appropriate for neuronal and melanocyte lineage differentiation. Methods: In this study, SSCs and hair follicle stem cells (HFSCs) were expanded in vitro by explant culture method and were compared in terms of proliferative potential and stemness; differentiation potential into melanocytes and neuronal lineage. Results: SSCs were found to be more proliferative in comparison to HFSCs, however, telomerase activity was more in HFSCs in comparison to SSCs. Capacity to differentiate into two lineages of ectoderm origin (neuronal and melanocyte) was found to be different. HFSCs cells showed more propensities towards melanocyte lineage, whereas SSCs were more inclined towards neuronal lineage. Interpretation & conclusions: The study showed that SSCs had differential advantage over the HFSCs for neuronal cell differentiation, whereas, the HFSCs were better source for melanocytic differentiation.

A Pilot, Randomized Sham Control Trial of Autologous Bone Marrow Derived Mononuclear Cells in Acute Ischemic Central Retinal Vein Occlusion.

In this pilot, sham controlled randomized control trial (RCT) in patients with ischemic central retinal vein occlusion (CRVO), we studied the safety and efficacy of intravitreal injection of autologous bone marrow derived mononuclear cells and found that both patients who received stem cell injections did not develop anterior segment neovascularization at 1 year follow up. Except for some sterile inflammatory reaction in the initial follow up, no long term injection related serious adverse events (SAEs) were observed. Based on our observations we recommend a larger, multicentric study to further establish the safety and efficacy of this treatment in patients with ischemic CRVO. Purpose: To study the safety and efficacy of autologous bone marrow derived mononuclear cells injected intravitreally in patients with ischemic CRVO. Study Design: Randomized sham controlled trial. Methods: 4 cases with ischemic CRVO were recruited into the study. 2 cases were randomized into intervention group and 2 into control group. Baseline investigations included best corrected visual acuity (BCVA), intra ocular pressure (IOP), fundus fluorescein angiography (FFA), gonioscopy and optical coherence tomography (OCT). Patients in the intervention group received intravitreal injection of autologous bone marrow derived mononuclear cells (MNCs) and those in control group received sham injection. Patients were followed up over a 12-month period. Main Outcome Measures: Development of anterior segment neovascularization. Results: Both patients in the intervention group did not develop anterior segment neovascularization over a follow up period of 12 months. 1 patient in control group developed neovascularization of iris and elevated intra ocular pressure over a follow up period of 6 weeks and required trabeculectomy for control of IOP. The other patient in control group was lost follow up after 2 weeks. Conclusions: Our initial observations suggest that intravitreal injection of mononuclear cells may reduce the risk of developing anterior segment neovascularization in patients with ischemic central retinal vein occlusion. A larger, multicentric study would be valuable to gain further evidence to our preliminary observations.

Culture & differentiation of mesenchymal stem cell into osteoblast on degradable biomedical composite scaffold: In vitro study.

Background & objectives: There is a significant bone tissue loss in patients from diseases and traumatic injury. The current autograft transplantation gold standard treatment has drawbacks, namely donor site morbidity and limited supply. The field of tissue engineering has emerged with a goal to provide alternative sources for transplantations to bridge this gap between the need and lack of bone graft. The aim of this study was to prepare biocomposite scaffolds based on chitosan (CHT), polycaprolactone (PCL) and hydroxyapatite (HAP) by freeze drying method and to assess the role of scaffolds in spatial organization, proliferation, and osteogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro, in order to achieve bone graft substitutes with improved physical-chemical and biological properties. Methods: Pure chitosan (100CHT) and composites (40CHT/HAP, 30CHT/HAP/PCL and 25CHT/HAP/PCL scaffolds containing 40, 30, 25 parts per hundred resin (phr) filler, respectively) in acetic acid were freeze dried and the porous foams were studied for physicochemical and in vitro biological properties. Results: Scanning electron microscope (SEM) images of the scaffolds showed porous microstructure (20-300 μm) with uniform pore distribution in all compositions. Materials were tested under compressive load in wet condition (using phosphate buffered saline at pH 7.4). The in vitro studies showed that all the scaffold compositions supported mesenchymal stem cell attachment, proliferation and differentiation as visible from SEM images, [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay, alkaline phosphatase (ALP) assay and quantitative reverse transcription (qRT)-PCR. Interpretation & conclusions: Scaffold composition 25CHT/HAP/PCL showed better biomechanical and osteoinductive properties as evident by mechanical test and alkaline phosphatase activity and osteoblast specific gene expression studies. This study suggests that this novel degradable 3D composite may have great potential to be used as scaffold in bone tissue engineering.

Efficacy of stem cell in improvement of left ventricular function in acute myocardial infarction - MI3 Trial.

Background & objectives: Acute myocardial infarction (AMI) is characterized by irreparable and irreversible loss of cardiac myocytes. Despite major advances in the management of AMI, a large number of patients are left with reduced left ventricular ejection fraction (LVEF), which is a major determinant of short and long term morbidity and mortality. A review of 33 randomized control trials has shown varying improvement in left ventricular (LV) function in patients receiving stem cells compared to standard medical therapy. Most trials had small sample size and were underpowered. This phase III prospective, open labelled, randomized multicenteric trial was undertaken to evaluate the efficacy in improving the LVEF over a period of six months, after injecting a predefined dose of 5-10 × 108 autologous mononuclear cells (MNC) by intra-coronary route, in patients, one to three weeks post ST elevation AMI, in addition to the standard medical therapy. Methods: In this phase III prospective, multicentric trial 250 patients with AMI were included and randomized into stem cell therapy (SCT) and non SCT groups. All patients were followed up for six months. Patients with AMI having left ventricular ejection fraction (LVEF) of 20-50 per cent were included and were randomized to receive intracoronary stem cell infusion after successfully completing percutaneous coronary intervention (PCI). Results: On intention-to-treat analysis the infusion of MNCs had no positive impact on LVEF improvement of ≥ 5 per cent. The improvement in LVEF after six months was 5.17 ± 8.90 per cent in non SCT group and 4.82 ± 10.32 per cent in SCT group. The adverse effects were comparable in both the groups. On post hoc analysis it was noted that the cell dose had a positive impact when infused in the dose of ≥ 5 X 108 (n=71). This benefit was noted upto three weeks post AMI. There were 38 trial deviates in the SCT group which was a limitation of the study. Interpretation & conclusions: Infusion of stem cells was found to have no benefit in ST elevation AMI. However, the procedure was safe. A possible benefit was seen when the predefined cell dose was administered which was noted upto three weeks post AMI, but this was not significant and needs confirmation by larger trials.

Autologous intravenous bone marrow mononuclear cell therapy for patients with subacute ischaemic stroke: a pilot study.

Background & objectives: Bone marrow mononuclear cell therapy has emerged as one of the option for the treatment of Stroke. Several preclinical studies have shown that the treatment with mononuclear cell (MNCs) can reduce the infarct size and improve the functional outcome. We evaluated the feasibility, safety and clinical outcome of administering bone marrow mononuclear cell (MNCs) intravenously to patients with subacute ischaemic stroke. Methods: In a non-randomized phase-I clinical study, 11 consecutive, eligible and consenting patients, aged 30-70 yr with ischaemic stroke involving anterior circulation within 7 to 30 days of onset of stroke were included. Bone marrow was aspirated from iliac crest and the harvested mononuclear cells were infused into antecubital vein. Outcomes measured for safety included immediate reactions after cell infusion and evidence of tumour formation at one year in whole body PET scan. Patients were followed at week 1, 4-6, 24 and 52 to determine clinical progress using National Institute of Health Stroke Scale (NIHSS), Barthel Index (BI), modified Rankin Scale (mRS), MRI, EEG and PET. Feasibility outcomes included target-dose feasibility. Favourable clinical outcome was defined as mRS score of 2 or less or BI score of 75 to 100 at six months after stem cell therapy. Results: Between September 2006 and April 2007, 11 patients were infused with bone-marrow mononuclear cells (mean 80 million with CD-34+ mean 0.92 million). Protocol was target-dose feasible in 9 patients (82%). FDG-PET scan at 24 and 52 wk in nine patients did not reveal evidence of tumour formation. Seven patients had favourable clinical outcome. Interpretation & conclusions: Intravenous bone marrow mononuclear cell therapy appears feasible and safe in patients with subacute ischaemic stroke. Further, a randomized controlled trial to examine its efficacy is being conducted.

Rapid multiplication and in vitro production of leaf biomass in Kaempferia galanga through tissue culture

An efficient protocol has been established for rapid multiplication and in vitro production of leaf biomass in Kaempferia galanga L, a rare medicinal plant. Different plant growth regulators like Benzyladenine (BA), Indoleacetic acid (IAA), Indolebutyric acid (IBA), Napthaleneacetic acid (NAA) and adenine sulphates (Ads) have been tried for induction of multiple shoots using lateral bud of rhizome as explants. The highest rate of shoot multiplication (11.5 +/- 0.6) shoot/explant as well as leaf biomass production (7.4 +/- 0.3) gram/explant was observed on Murashige and Skoog medium supplemented with Benzyladenine (1 mg/l) and Indoleacetic acid (0.5 mg/l). Data of shoot multiplication and leaf biomass production were statistically analysed. Upon excission of leaves after 2 months of culture under sterile condition, the base of each plantlet was transferred to fresh media which could produce the same leaf biomass within another 2 months in a 50 ml culture tube containing 20 ml and 250 ml conical flasks containing 30 ml Murashige and Skoog medium. The rate of multiplication and leaf biomass production remained unchanged in subsequent subcultures. The regenerated plantlets were acclimatized in greenhouse and subsequently transferred to the field. Survival rate of the plantlets under ex vitro condition was 95 percent. Genetic fidelity of the regenerants was confirmed using random amplified polymorphic DNA (RAPD) marker. The protocol could be commercially utilized for large scale production of true-to-type plantlets and as an alternative method of leaf biomass production in Kaempferia galanga.

In silico characterization of genetic homology in nuclear-encoded apicoplast-targeted genes between Plasmodium falciparum & P. vivax.

Background & objectives: Resistance to anti-malarial drugs by the parasites is one of the major obstacles to malaria control. The primary objective of this work was to fi nd specifi c nuclear-encoded-apicoplasttargeted genes that are conserved between two different human malaria parasite species, Plasmodium falciparum and P. vivax to fi to fi nd a common drug/vaccine targets for both the species. Methods: Using computational genomics, possible nuclear-encoded-apicoplast-targeted genes were identifi ed in P. falciparum genome. With comparative genomic approaches, homologous genes were identifi ed between the two different human malaria species, P. falciparum and P. vivax. Results: Of the total 545 reported nuclear-encoded-apicoplast-targeted genes in P. falciparum, we could narrow down to as less as fi ve genes that were found to have highly conserved nucleotide stretches in P. vivax. However, two such genes were of importance, as the majority of the protein coding regions (exons) of these genes were found to be highly conserved between them. Interpretation & conclusion: This preliminary study shows that nuclear-encoded-apicoplast-targeted genes were conserved between the two human malaria parasites and these could be targeted for developing a common drug to cure both forms of malaria.

Fine-scale genetic characterization of Plasmodium falciparum chromosome 7 encompassing the antigenic var and the drug-resistant pfcrt genes.

The fact that malaria is still an uncontrolled disease is reflected by the genetic organization of the parasite genome. Efforts to curb malaria should begin with proper understanding of the mechanism by which the parasites evade human immune system and evolve resistance to different antimalarial drugs. We have initiated such a study and presented herewith the results from the in silico understanding of a seventh chromosomal region of the malarial parasite Plasmodium falciparum encompassing the antigenic var genes (coding pfemp1) and the drug-resistant gene pfcrt located at a specified region of the chromosome 7. We found 60 genes of various functions and lengths, majority (61.67%) of them were performing known functions. Almost all the genes have orthologs in other four species of Plasmodium, of which P. chabaudi seems to be the closest to P. falciparum. However, only two genes were found to be paralogous. Interestingly, the drug-resistant gene, pfcrt was found to be surrounded by seven genes coding for several CG proteins out of which six were reported to be responsible for providing drug resistance to P. vivax. The intergenic regions, in this specified region were generally large in size, majority (73%) of them were of more than 500 nucleotide bp length. We also designed primers for amplification of 21 noncoding DNA fragments in the whole region for estimating genetic diversity and inferring the evolutionary history of this region of P. falciparum genome.

Abdominal pigmentation and growth temperature in Indian Drosophila melanogaster : Evidence for genotype-environment interaction.

Phenotypic variability for abdominal pigmentation in females of an Indian natural population of Drosophila melanogaster was studied using isofemale lines and by rearing the larvae and pupae at 4 different temperatures ranging from 20–30°C. The dark pigmented area was found to increase in all the three segments when the growth temperature decreases. A significant positive correlation was detected for the occurrence of dark pigmentation in the 5th and 6th segments in each growth temperature but for other comparisons the correlation was not regular. Analysis of variance (ANOVA) was carried out both for individual segments over different growth temperatures and also for each temperature over the three abdominal segments and in all cases found to be statistically significant. The results are quite different from the earlier observation in French Drosophila melanogaster and suggest that genes controlling pigmentation are temperature dependent; temperature could affect post-transitional events involved in pigmentation. The present findings also clearly indicate that significant genotype-environment interaction exists, responsible for the production of desired phenotype at the opportune moment during the life span of a species.