Evaluation of Bone Marrow Processing Protocol for Therapeutic Applications via Culture and Characterization of Mesenchymal Stem Cells.

INTRODUCTION: Human mesenchymal stem cells from bone marrow (hMSCs) have broad therapeutic potential. These cells can be are readily isolated from bone marrow by their property to adhere to tissue culture treated culture wares. However, the proliferation rates and other properties of the cells gradually change during expansion. This study aims to validate the protocol of isolation and differentiation of hMSCs from bone marrow for therapeutic applications. METHODS: Sixty ml of bone marrow was extracted from 5 patients and MSCs were isolated. These were characterized by Flow Cytometry, CFU assay and were differentiated into bone, fat cells and neurocytes. RESULTS: The cells were having healthy morphology. These were positive for the markers CD105, CD90 and CD73 and negative for CD45, CD34 and HLA-DR. The cells could differentiate into fat, bone and neural cells. CONCLUSION: MSCs from the bone marrow were isolated and differentiated. These cells were morphologically healthy and passed CFU assay. The cells exhibited differentiation potential into bone, fat and neural tissue. These cells can be used in therapeutic applications.

A Short Study Report on Bone Marrow Aspirate Concentrate Cell Therapy in Ten South Asian Indian Patients with Autism.

Autism is a neural disorder presenting in the early developmental period, usually in the first 2 years of life. It is characterized by persistent deficits in social communication and social interaction, restricted and repetitive patterns of behavior, interests, or activities, and causes clinically significant impairment in social, occupational, or other important areas of functioning.Cellular therapy is an advanced approach to treat disorders where current therapies do not offer a cure or efficient symptom relief. Herein we present the data of ten autistic patients who were enrolled in this study utilizing stem cells. All patients were scored on ISAA and WeeFIM scales before and at 3, 6, 12 or 24 months following the injection of stem cells. The ISAA scores of the patients improved with the injection, and no adverse effects were noted. We report promising results in this small pilot study using autologous Bone Marrow Aspirate Concentrate (BMAC) injected intrathecally into these ten enrolled autistic patients.

Therapy with Bone Marrow-Derived Autologous Adult Stem Cells in Quadriparesis due to Motor Neuron Disease.

OBJECTIVE: To report the safety and therapeutic effectiveness of application of concentrated bone marrow aspirate in three bedridden patients with weakness in both legs, and monitor potential improvement in neurological outcomes. DESIGN: Case report. Intervention: Five infusions of 3x108 mononuclear cells were administrated with 12 week intervals. Bone marrow (240ML) were obtained from the posterior superior iliac spine and Bone marrow mononuclear cells were enriched by standard manual close method under aseptic condition. RESULTS: During the follow-up study of one year after stem cell implantation, the conditions of all three patients were improved and were confirmed by physical assessment, muscle charting and Electromyography (EMG). One year after stem cell implantation patients who were bedridden before treatment could sit without support and walk with support up to 200 feet at a stretch. CONCLUSION: The local application of a cocktail of regenerative cell population found in an MNC fraction of bone marrow was safe and effective in improving quality of life and muscle strength in ALS patients. This case opens the need for further investigations on Autogenic stem cell transplant therapies for MND disease.

Administration of Autologous Bone Marrow-Derived Stem Cells for Treatment of Cerebral Palsy Patients: A Proof of Concept.

BACKGROUND: Stem cell therapy is a promising treatment for cerebral palsy, which refers to a category of brain diseases that are associated with chronic motor disability in children. Autologous bone marrow stem cells may be a better cell source and have been studied for the treatment of cerebral palsy because of their functions in tissue repair and the regulation of immunological processes. METHODS: To assess autologous marrow stem cells as a novel treatment for patients with moderate-to-severe cerebral palsy, a total of 10 cerebral palsy patients were enrolled in this clinical study with 24 months follow-up. A total of 10 cerebral palsy patients received autologous bone marrow cells transplantation (4.5 × 108 mononuclear cells; 90% viability) into the subarachnoid cavity and rehabilitation. RESULTS: We recorded the gross motor function measurement scores, manual ability function measurement score, and adverse events up to 24 months post-treatment. The gross motor function measurement scores were significantly higher at month 6 post-treatment compared with the baseline scores and were stable up to 24 months follow-up. The increase in manual ability and communication function measurement scores at 6 months were not significant when compared to the baseline score. All the 10 patients survived and none of the patients experienced any serious adverse events or complications. CONCLUSION: Our results indicated that bone marrow derived MNCs are safe and effective for the treatment of motor deficits related to cerebral palsy. Further randomized clinical trials are necessary to establish the efficacy of this procedure.

Autologous Bone Marrow-Derived Stem Cells in Spinal Cord Injury.

INTRODUCTION: Spinal cord injury is a traumatic neurological condition which makes the patient disable. Its management still remains challenging but advancements in the regenerative medicine have changed the approach of treating this serious debilitating condition of the central nervous system. Cell based therapies can restore function in spinal cord injury by replacing the lost neural tissue. These therapies also rejuvenate the existing intact neurons by facilitating remyelination and by repairing and reducing progressive tissue damage and scarring. METHODS: Autologous bone marrow stem cells were collected from the patients. 5 ml of the processed sample was injected back into the patients via lumbar puncture at L1/L2 level. The bone marrow harvesting and administration was repeated every 4 weeks 3 times (12 weeks). RESULTS: Significant improvements were noticed following the injections into the patients with the duration of injury less than 6 months. ASIA grade improvements were observed in 6 out of 10 patients. VTC and walking, at least with the support, was restored in eight patients. Bladder control and sexual functions improved in three and five patients respectively. Eight patients exhibited decreased spasticity. DISCUSSION: We believe that autologous bone marrow stem cells contributed towards the neuroplaticity and/or paracrine effect due to which we observed the considerable improvements in the conditions of the patients. CONCLUSION: This preliminary proof of patient improvement reinforces the potential of autologous bone marrow stem cell treatment in the patients suffering from Spinal Cord Injury. Although the results are encouraging further studies are needed to substantiate the claims.

Evidence for extended age dependent maternal immunity in infected children: mother to child transmission of HIV infection and potential interventions including sulfatides of the human fetal adnexa and complementary or alternative medicines.

The two neighboring southwestern states of India, Karnataka and Maharashtra, have high incidence of HIV/AIDS and are among the six most high prevalence HIV infected states. In Karnataka state, the northern districts of Bagalkot, Belgaum and Bijapur (the three Bs) and in Maharashtra state, the southern districts of Sangli, Satara, and Solapur (the three Ss) are the areas with the highest incidence of HIV/AIDS. We have evaluated the incidence of maternal to child transmission (MTCT) of HIV-1 infection in Belgaum District which is more than 500 kilometers distance by road from the campus in greater Bangalore (Karnataka State). We have obtained the prenatal CD4 counts of HIV infected pregnant mothers. We have also screened the HIV infected children in two orphanages (rehabilitation centres for HIV infected children) in Belgaum District. The clinical conditions of these infected children were assessed for their CD4 counts, anti-retroviral therapy (ART) intake status, outpatient illnesses and body composition. We have observed that there is an influence of the age factor on the CD4 counts of the HIV infected children. Further, in view of the role of our recently found involvement of sulfatide, 3-O- galactosylceramide, in inhibition of HIV-1 replication and enhancement of hematopoiesis which is otherwise inhibited due to such infection, we have discussed the possible role of sulfatides that biologically occur in the fetal adnexa (placentatrophoblasts /amnion/chorion-umbilical cord), in containing HIV infection as a potential safer alternative to the ART regimens currently approved to be clinically practiced. Lastly, we have discussed the complementary and alternative medicine (CAM) therapies such as evidence based yoga and ayurveda as add-on to ART in potential elimination of MTCT of HIV infection. Out of a total of 150 children delivered by HIV infected mothers, 13 children were found to be positive as determined by the dried blood smear (DBS) for virological testing, giving an incidence of about 8.66% in the Belgaum district during the last two years, in spite of the prescription of currently available ART regimens. All the 13 HIV-transmitting mothers had normal vaginal deliveries. Though 12% of the total 150 deliveries required lower segment caesarean section (LSCS), none among them resulted in MTCT of HIV. Comparison of the prenatal CD4 counts between transmitting and non-transmitting mothers did not show significant differences (p=0.25) thus suggesting indirectly that HIV-1 proviral loads (undetermined / unavailable) need not necessarily determine the fate of incidence of vertical transmission. The mean age of 44 HIV infected children (14 females, 30 males) that were screened in two orphanages was 10.8±3.1 years. Out of these 44 children, 27 were taking ART (61.36%) with mean duration of consumption being 2.8±2.28 years. Fifty percent (n=22) of the children were suffering from at least one outpatient illness, out of which 13 were taking ART. Their mean basal metabolic rate (BMR), body mass index (BMI), muscle mass, fat mass and fat % were 795.45±106.9, 14.55±1.9 kg/m(2), 9.54±3.4 kg, 3.69±2.24 kg and 15.04±7.8% respectively. Comparison between the children taking ART (on-ART, n=27) and those not taking ART (non-ART, n= 17) showed that though there was no significant difference in the average age of the two groups, on-ART children had significantly higher BMR (p=0.05), and muscle mass (p=0.004), than non-ART. The CD4 counts, BMI, fat mass and fat percentage did not show significant statistical differences between the two groups. The CD4 counts of the children (both on-ART and non-ART) of age 8 years and below (n=12) were found to be significantly higher (p=0.04) than those of age 14 and above (n=10). All the children in age group of 14 years and above (n=10) except one child were on ART, whereas 7 out of 12 children in age group of 8 years and below were on-ART. In one of the rehabilitation centers called Aadhar, among non-ART children, a significant correlation was observed between the age of the child and CD4 counts (measured separately in the months of June 2011 and December 2011). Both the CD4 counts measured in June 2011 (n=6; r=-0.82, p= 0.04) as well as in December 2011 (n=6; r=-0.97, p=0.001) showed a significant decline as the age progressed. Also, at the same center, among on-ART children, the CD4 counts in June 2011 (n=7) and December 2011 (n=8) were significantly different between the children in the age group of 8 below years, and those in the age group of 14 years and above (p= 0.005). As HIV infected children grow in age, they may lose maternal derived immunity as shown by the decrease in CD4 counts, irrespective of their ART status. It is to be expected from these results that the conferred maternal immunity (possibly primarily humoral and secondarily cytotoxic immune responses) to the virus acquired at child birth taper off and eventually overcome by the generation of mutant HIV strains in the children, as the life spans of the infected children progress. We have discussed safer therapeutic interventions whose efficacy on HIV/AIDS may be synergistic to or even substitute the existing treatment strategies. Some of such interventions may even be customized to help eliminate MTCT. Further, these virus infected pregnant mother patient blood / serum samples could prove useful in the vaccine development against HIV infection.

Peripheral CD300a+CD8+ T lymphocytes with a distinct cytotoxic molecular signature increase in pregnant women with chronic chorioamnionitis.

PROBLEM: CD300a is an immunomodulatory molecule of the immunoglobulin receptor superfamily expressed in the leukocytes of myeloid and lymphoid lineages. However, its biological function on CD8+ T lymphocytes remains largely unknown. This study was conducted to assess the biological significance of CD300a expression in T lymphocytes and to determine whether its expression in peripheral T lymphocytes changes in pregnant women presenting with antifetal rejection. METHODS OF STUDY: Microarray analysis was performed using total RNA isolated from peripheral CD300a+ and CD300a- T lymphocytes. Flow cytometric analysis of the peripheral blood samples of pregnant women and pathologic examination of the placentas were conducted. RESULTS: A large number of genes (N = 1245) were differentially expressed between CD300a- and CD300a+ subsets of CD8+ T lymphocytes, which included CCR7, CD244, CX3CR1, GLNY, GZMB, GZMK, IL15, ITGB1, KLRG1, PRF1, and SLAMF7. Gene ontology analysis of differentially expressed genes demonstrated enrichment of biological processes such as immune response, cell death, and signal transduction. CD300a expression in CD8+ T lymphocytes was coupled to a more cytotoxic molecular signature. Of note, the proportion of CD300a+CD8+ T lymphocytes increased in pregnant women with chronic chorioamnionitis (antifetal rejection of the chorioamniotic membranes; P < 0.05). CONCLUSION: The findings of this study strongly suggest an increase in systemic T-lymphocyte-mediated cytotoxicity in pregnant women with chronic chorioamnionitis as a manifestation of maternal antifetal rejection.

Sulfatide--a new candidate for ART treatment in HIV-1 infection.

New combination drug treatment(s) now available to patients with HIV-1 infection allows them to live longer lives with good quality of life although they suffer from the incurable HIV-1 infection. In a previous study we found that sulfatide was efficient in lowering HIV-1 viral loads in SCID mice engrafted with human fetal liver/thymus tissues (SCID-hu). Current antiviral treatments carry an increased risk of other complications like cardiovascular disease and diabetes after long-term use. There is a need for new potent safe pharmaceutical agents. Endogenous sulfatide is a mixture of -isoforms, i.e. sulfatide molecules with different long-chain bases and fatty acid chain lengths and saturation. Sulfatide isoforms may have different physicochemical properties i.e, they are of different potency at different target cells. Other investigators have shown that incubation of cultured cells with sulfatide incorporated into the plasma membrane inhibited HIV-1 entry into the cells thereby inhibiting intracellular HIV-1 replication. We have shown that CD1d dependent stimulation by sulfatide may activate pDC antigen expressing cells that produce type I inteferons. Type I inteferons are known to reduce HIV-1 replication. This could provide a second likely explanation (after the inhibition of virus entry) for the more efficient lowering of HIV-1 viral loads in sulfatide versus AZT treated mice. This review aims to show the efficiency of sulfatide in reducing HIV-1 viral loads as compared to conventional HAART treatment. We also discuss the risks of HAART treatment and propose a clinical alternative of sulfatide in HIV-1 infection.

Sulfatide administration leads to inhibition of HIV-1 replication and enhanced hematopoeisis.

The pathogenesis of HIV-1 infection is a complex process in which Natural Killer (NK) and Natural Killer T (NKT) cells play an important role. NKT cells express markers for NK cells and a TCR of the conventional T cells and recognize lipid antigens presented by the non-polymorphic CD1 molecule. CD1d-restricted type I NKT cells express an invariant TCR and can recognize αGalCer, whereas a major subset of type II NKT expressing diverse TCR can recognize a self-glycolipid, sulfatide. It has been shown that CD4+ type I NKT cells are infected by HIV-1 and decreased in HIV-1-infected individuals. However, their exact role in HIV-1 infection as well as the biology and function of the type II NKT cell subset in HIV-1 infection and disease progression are not known. Our earlier studies have shown that activation of CD1d-restricted type II NKT cells by sulfatide and their interactions with plasmacytoid dendritic (pDC) and myeloid dendritic (mDC) cells result in anergy induction in type I NKT cells in several models. Here we used SCID-Hu (Thy/Liv) animals, co-implanted with human fetal liver and thymus, and found that these implants contain both type I and type II NKT cells, CD161+CD3+ NKT cells, NK cells and dendritic cells during HIV-infection. We found that the administration of sulfatide (bi-weekly, 20 µg/animal, i.p.) in SCID-Hu animals inhibits HIV-1 replication more efficiently than treatment with the nucleoside analog reverse transcriptase inhibitor, AZT. Virus replication was lowered significantly up to 4-8 weeks post infection. Furthermore sulfatide administration also resulted in significant retention of hematopoeisis that is lost during HIV-1 infection. Advantageously, sulfatide administration itself was not associated with anemia or bone marrow suppression, that are severe side effects of HAART. Since the CD1d-mediated immune pathway is highly conserved between rodents and humans, sulfatide treatment may represent a novel HLA-independent approach for intervention of HIV-1 pathogenesis.

Placental membrane as a source of mesenchymal stem cells.

Non-embryonic stem cells have the potential for being used in therapeutic attempts to develop treatments for human diseases and injuries. There is an increasing demand for non invasive treatments for a number of diseases and disorders that strike and disable relatively young people that would otherwise have many active years ahead. As longevity increases and we face new challenges in treating patients who suffers from disorders that reduces their quality of life already at middle age. Simple, secure methods to isolate non-embryonic stem cells from different tissues is one step towards making those treatments available to people suffering from Parkinson's disease, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis, and rheumatoid arthritis.

Rescue of multi-lineage hematopoiesis during HIV-1 infection by human c-mpl gene transfer and reconstitution of CD34+ progenitor cells in vivo.

Cytopenias arising from hematopoietic abnormalities are a severe common complication contributing to early mortality in HIV/AIDS patients. The proto-oncogene c-mpl, identified as the thrombopoietin receptor is involved in multilineage differentiation of CD34+ hematopoietic progenitor cells. We have introduced the c-mpl gene into CD34+ cells via transduction of the lentivirus p156RRLsinPPTmPGK-CMPL-PRE. The lentiviral construct expresses c-mpl on approximately 90% of purified CD34+ cells. These transduced cells have then been reconstituted into human fetal thymus/liver implants in severe combined immunodeficient mice (SCID-hu Thy/Liv). The c-mpl expression on transduced CD34+ cells is not susceptible to downregulation due to the effects of HIV-1 infection. Reconstituted CD34+ cells transduced with control lentivirus, p156RRLsinPPTmPGK-EGFP-PRE, express EGFP at > 90%. Reconstituted c-mpl expressing SCID-hu implants show almost maximum rescue (approximately 90%) of myelopoiesis, erythropoiesis and megakaryopoiesis, during HIV-1 infection in vivo, at 6 weeks post-infection. We also show that the differentiated multi-lineage progeny colonies and thymocytes in mice reconstituted with the c-mpl transduced CD34+ cells, carry the HLA Class I loci phenotypes of these donor cells, in the implants of the recipient SCID-hu animals. We propose a gene therapeutic strategy, with c-mpl as the major genetic component, to address the morbidity and mortality resulting from cytopenias in HIV infected patients.

Thrombocytopenia in HIV infection: impairment of platelet formation and loss correlates with increased c-Mpl and ligand thrombopoietin expression.

Thrombocytopenia is a common hematologic disorder in patients infected with the human immunodeficiency virus (HIV) and represents a risk for bleeding which is further deleterious during surgery. The major causes of the thrombocytopenia include accelerated peripheral platelet destruction by antiplatelet antibodies and insufficient production of platelets from the infected megakaryocytes. Additionally, at an earlier stage of platelet development, HIV may inhibit megakaryopoiesis at multiple stages of pluripotent CD34+ progenitor stem cell differentiation possibly contributing to decreased levels of platelets in circulation. In HIV infected patients, both the serum thrombopoietin (TPO) levels and theTPO-c-Mpl complexes on the platelet surface were significantly elevated. Therapeutic infusion of HIV infected patients with pegylated recombinant human megakaryocyte growth development factor (PEG-rHu-MGDF) restores platelet counts to normal levels and reduces the c-Mpl expression per platelet. In vitro aggregation of platelets treated with TPO and agonist, adenosine diphosphate (ADP), decrease the dose of ADP that is required for half-maximum aggregation. In vivo dosing does not effect platelet aggregation showing that the metabolism of TPO following its internalization through TPO-c-Mpl complex is rapid and that dosing within the therapeutic range does not constitute increased risk of thrombotic disease.

Chimeric SCID-hu Model as a Human Hematopoietic Stem Cell Host That Recapitulates the Effects of HIV-1 on Bone Marrow Progenitors in Infected Patients.

The chimeric small animal model system wherein the severe combined immunodeficient mouse is transplanted with human fetal thymus and liver tissues (SCID-hu Thy/Liv) has been used in different areas of experimental research [1]. A living model system offers the advantage that studies of longer duration than in vitro can be performed. Here we discuss the importance of experimental in vivo studies and how well the data generated using this model system correlates with epidemiological studies from HIV-positive humans. Patients with HIV-1 infection often suffer from cytopenias, with thrombocytopenia typically having an earlier onset than anemia and neutropenia. The cytopenias in patients may be caused by virus effects at the stem cell level, or the mature blood cell level, or by other factors like medications used to treat the infection. Several studies preformed in SCID-hu animals show that the function of human hematopoietic progenitor stem cells are indirectly affected by HIV-1 infection. Colony forming activity (CFA) assays on hematopoietic progenitor cells derived from SCID-hu animals and human bone marrow have been performed as a measure of progenitor cellular function, and HIV-1 infection decreased CFA in multiple lineages that include megakaryoid, erythroid and myeloid types. Highly active antiretroviral therapy (HAART) partially and transiently reduces the inhibition of multilineage CFA and data suggest that HIV-1 indirectly affects stem cell differentiation into multiple lineages. The cojoint human hematopoietic organ of this small animal model system recapitulates the inhibitory effects of HIV-1 infection on the function of progenitor cells of the human bone marrow. Thus the SCID-hu (Thy/Liv) model is a useful in vivo system for preclinical translational research towards development of stem cell therapies in HIV infection.

Expression of adducin genes during erythropoiesis: a novel erythroid promoter for ADD2.

OBJECTIVE: The first objective of this study was to examine the differences in levels of adducin (ADD1, ADD2, ADD3) mRNA expression during human erythropoiesis. The second objective was to determine whether the rapid induction of ADD2 expression could be attributed to a novel erythroid-specific promoter. METHODS: Expression of mRNA was quantified using real-time RT-PCR. Primary erythroid precursors were isolated from normal human bone marrow using fluorescence-activated cell sorting. Two model systems were compared: CD34(+) hematopoietic stem cells induced to differentiate with erythropoietin and HEL cells induced to differentiate with hemin. 5'RACE analysis was performed using primary human erythroblasts as starting material. RESULTS: All three adducin genes showed different patterns of expression during erythropoietic differentiation of cultured CD34(+) stem cells. Levels of ADD3 mRNA were higher than levels of ADD2 mRNA at early stages of erythropoiesis. Expression of ADD2 was induced to very high levels (100 times baseline) in erythropoietin-stimulated cultures. 5'RACE analysis identified a novel starting exon and putative erythroid promoter for ADD2. CONCLUSION: These results suggest that expression of each adducin gene is regulated in a gene-specific manner during erythropoiesis. The early expression of ADD3 suggests that it may have a role in erythroblasts but is replaced by ADD2 in later stages of erythropoiesis. The very high levels of expression of ADD2 suggest that its promoter may be useful for directing erythroid-specific gene expression.

The role of phospholipases A2 in the stimulation of neutrophil motility by cobra venoms.

Neutrophil (PMN) accumulation frequently occurs at the site of snakebite as part of the inflammatory response to envenoming. We demonstrate here that the venoms of the cobras, Naja naja and N. mossambica, and two purified venom phospholipase A(2)s (PLA(2)s) isolated from the latter venom, stimulate CD11b translocation from the PMN granule store to the plasma membrane and enhance neutrophil motility on collagen-coated surfaces. These effects were partially attenuated by the PLA(2) inhibitor, aristolochic acid, and almost completely abolished by the specific cytosolic PLA(2) inhibitor, methylarachidonylfluorophosphonate (MAFP). Annexin V and inhibitors of collagenase, cyclo-oxygenase and lipo-oxygenase, all inhibited PMN motility to a variable extent. FACS analysis and confocal microscopy showed that Annexin V interfered with binding and rapid endocytosis of the venom PLA(2). These results indicate that venom and venom PLA(2) preparations first caused a non-enzymatic stimulation of PMN leading to the activation of cytosolic PMN PLA(2) and production of arachidonate metabolites involved in stimulation of PMN degranulation and motility. The evidence suggests that venom PLA(2) then interacts with anionic phospholipids exposed on stimulated PMN, becomes endocytosed, and then contributes itself to the production of chemoattractants responsible for PMN accumulation at the site of the snakebite.