Development of an alginate-chitosan biopolymer composite with dECM bioink additive for organ-on-a-chip articular cartilage.

In vitro use of articular cartilage on an organ-on-a-chip (OOAC) via microfluidics is challenging owing to the dense extracellular matrix (ECM) composed of numerous protein moieties and few chondrocytes, which has limited proliferation potential and microscale translation. Hence, this study proposes a novel approach for using a combination of biopolymers and decellularised ECM (dECM) as a bioink additive in the development of scalable OOAC using a microfluidic platform. The bioink was tested with native chondrocytes and mesenchymal stem cell-induced chondrocytes using biopolymers of alginate and chitosan composite hydrogels. Two-dimensional (2D) and three-dimensional (3D) biomimetic tissue construction approaches have been used to characterise the morphology and cellular marker expression (by histology and confocal laser scanning microscopy), viability (cell viability dye using flow cytometry), and genotypic expression of ECM-specific markers (by quantitative PCR). The results demonstrated that the bioink had a significant impact on the increase in phenotypic and genotypic expression, with a statistical significance level of p < 0.05 according to Student's t-test. The use of a cell-laden biopolymer as a bioink optimised the niche conditions for obtaining hyaline-type cartilage under culture conditions, paving the way for testing mechano-responsive properties and translating these findings to a cartilage-on-a-chip microfluidics system.

Standardizing Chondrocyte Isolation and Articular Cartilage Decellularization: A Versatile Bioink for Tissue Engineering Applications.

The extracellular matrix (ECM) is a noncellular component of tissues that provides structural and biochemical support to cells. The purpose of decellularization is to provide a tissue-specific niche to preserve the architecture, composition, and signaling molecules of the ECM. The current protocol discusses the standardization of chondrocyte isolation and the preparation of acellular ECM as a bioink additive from human native articular cartilage. Isolated chondrocytes with bioink additives provide a tissue-specific microenvironment. Herein, we discuss a standardized protocol with multiple applications in the area of organ-on-a-chip model development, spheroid formation, microfluidics platform, bioprinting, and tissue engineering. Cartilage tissue engineering is complex owing to the heterogeneous complex proteins, which are a challenge to synthesize; hence, this protocol in many ways offers cues to exploit the acellular ECM for multiple ongoing research studies.

Extracellular matrix composition analysis of human articular cartilage for the development of organ-on-a-chip.

INTRODUCTION: Articular cartilage has a complex extracellular matrix (ECM) that provides it a defined architecture for its load-bearing properties. The complete understanding of ECM components is imperative for developing biomimetic organ-on-a-chip tissue construct. OBJECTIVE: This study aimed to decellularize and characterize the ECM for its protein profiling to generate a niche for enhanced chondrocyte proliferation. METHODS: Articular cartilage scrapings were subjected to mechanical and collagenase digestion, followed by sodium dodecyl sulfate (SDS) treatment for 8 h and 16 h. The de-cellularization efficiency was confirmed by hematoxylin & eosin, alcian blue, masson's trichrome staining, and scanning electron microscopy (SEM). The ECM protein profile was quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS) using a bottom-up approach. RESULTS: Histological characterization revealed void lacunae that lacked staining for cellular components. The ECM, sulfated glycosaminoglycan content, and collagen fibers were preserved after 8 h and 16 h of de-cellularization. The SEM ultrastructure images showed that few chondrocytes adhered to the ECM after 8 h and cell-free ECM after 16 h of de-cellularization. LC-MS/MS analysis identified 66 proteins with heterotypic collagen types COL1A1-COL6A1, COL14A1, COL22A1 and COL25A1 showed moderate fold change and expression levels, while COL18A1, COL26A1, chondroitin sulfate, matrix metalloproteinase-9 (MMP9), fibronectin, platelet glycoprotein 1 beta alpha (GP1BA), vimentin, bone morphogenetic protein 6 (BMP6), fibroblast growth factor 4 (FGF4) and growth hormone receptor (GHR) showed maximum fold change and expression levels. CONCLUSIONS: The standardized de-cellularization process could preserve majority of ECM components, providing structural integrity and architecture to the ECM. The Identified proteins quantified for their expression levels provided insight into engineering the ECM composition for developing cartilage-on-a-chip.

Effect of engineered superparamagnetic iron oxide nanoparticles in targeted cardiac precursor cell delivery by MRI.

A scientific approach is presented describing the fabrication of nanoprobe (GloTrack) that can act as cardiac precursor label to segregate cells from cardiac/non cardiac origins and traced by magnetic resonance imaging (MRI). Signal regulatory protein alpha (SIRPA) and kinase domain receptor (KDR) recognizing antibodies, form a layer on super paramagnetic iron oxide nanoparticle - poly-ethylene glycol (SPION-PEG) complex, and bind to protein expressed on the surface of cardiac muscle cells. Physical attributes size, distribution, labelling efficiency, echocardiogram (ECG) changes and bio-distribution by MRI were analysed. The results indicate that GloTrack has an average size of 471 nm, exhibits negative potential and promotes labelling efficiency. The bio-distribution of GloTrack in in vivo experiments was traceable in 7T MRI showing high accumulation of GloTrack in cardiac muscles as compared to the liver and spleen. ECG data revealed that GloTrack segregated cardiac precursors has the potential benefit in treating heart failure, thereby paving way in the development of minimal cell manipulation with targeted cell delivery approaches.

Distinct cell death markers identified in critical care patient survivors diagnosed with sepsis.

Sepsis is an abnormal immune response to infection characterized by an overwhelming systemic inflammation and cell death. Non-apoptotic cell death pertaining to pyroptosis, necroptosis and autophagy contribute to sepsis pathogenesis apart from classical apoptotic cell death. The objective of the current study is to investigate the presence of molecular markers of relevance to apoptotic and non-apoptotic cell death in control healthy subjects and septic patient survivors. Sepsis survivors (N = 24) and healthy human volunteers (N = 16) [40 total subjects] were recruited into the study. Clinical intervention included antibiotic treatment regimen administered to patients upon clinical diagnosis of sepsis followed by blood draw 18-24 hr post-antibiotic dose. Serum samples analyzed by enzyme-linked immunosorbent assay (ELISA) and peripheral blood mononuclear cells (PBMCs) by flow cytometry analysis for identification of cell death markers. Cell death markers analyzed by ELISA and flow cytometry included caspase-1, caspase-3, MLKL, RIPK3, p62 and LC3B. Serum and peripheral blood mononuclear cells (PBMCs) of septic survivors and healthy controls analyzed for the presence of distinct cell death markers. Markers of relevance to apoptosis (caspase-3), pyroptosis (caspase-1), necroptosis (MLKL) and autophagy (p62 and LC3B) were compared between septic survivors and healthy controls. ELISA analysis suggested significant alterations in the serum levels of non-apoptotic cell death markers, caspase-1 and p62/SQSTM1, in septic survivors compared to healthy controls (p < 0.05). There was no significant difference in the serum levels of caspase-3 and MLKL between septic survivors and healthy control subjects (p> 0.05). Intracellular caspase-1 levels did not show any significant alterations between septic survivors and healthy control subjects (p > 0.05). Flow cytometry analysis suggested significant increase in the intracellular expression of caspase-3, MLKL and its associated kinase RIPK3, and p62/SQSTM1 (p < 0.05) in sepsis patient survivors when compared to healthy human subjects. The current observational study identified significantly elevated levels of non-apoptotic cell death markers in sepsis patients compared to healthy controls. Noteworthy observation is the significant modulation of non-apoptotic cell death markers in serum samples derived from septic survivors post-antibiotic administration compared to healthy control subjects. Preliminary results serve as a basis for further mechanistic investigations to elucidate the role of distinct cell death markers in the prediction of clinical outcomes in sepsis.

Development of Bioengineered Organ Using Biological Acellular Rat Liver Scaffold and Hepatocytes.

The increasing demand for organs for transplantation necessitates the development of substitutes to meet the structural and physiological functions. Tissue decellularization and recellularization aids in retaining the three-dimensional integrity, biochemical composition, tissue ultra-structure, and mechanical behavior, which makes them functionally suitable for organ transplantation. Herein, we attempted to rebuild functional liver grafts in small animal model (Wistar rat) with a potential of translation. A soft approach was adopted using 0.1% SDS (Sodium Dodecyl Sulfate) for decellularization and primary hepatocytes were used as a potential cell source for recellularization. The decellularization process was evaluated and confirmed using histology, DNA content, ultra-structure analysis. The resultant scaffold was re-seeded with the rat hepatocytes and their biocompatibility was assessed by its metabolic functions and gene expression. The structural components of the Extracellular matrix (ECM) (Laminins, Collagen type I, Reticulins) were conserved and the liver cell-specific proteins like CK-18, alpha-fetoprotein, albumin were expressed in the recellularized scaffold. The functionality and metabolic activity of the repopulated scaffold were evident from the albumin and urea production. Expression of Cytokeratin-19 (CK-19), Glucose 6-Phosphatase (G6P), Albumin, Gamma Glutamyl Transferase (GGT) genes has distinctly confirmed the translational signals after the repopulation process. Our study clearly elucidates that the native extracellular matrix of rat liver can be utilized as a scaffold for effective recellularization for whole organ regeneration.

Standardization and quality assessment for clinical grade mesenchymal stem cells from human adipose tissue

Abstract Background Mesenchymal stem cells have immense potential in stem cell-based therapies, however there is a pre-requisite to develop a curative cell dose. Adipose tissue-derived mesenchymal stem cells are promising mainly due to their potential abundance, immunomodulatory effect and remarkable differentiation potential. Nevertheless, senescence may develop during their in vitro expansion due to the incidence of genetic instability. Hence, it is important to attain an ideal balance between mesenchymal stem cell growth, quality and genetic integrity before their clinical use. Methods Stromal vascular fraction was obtained from omentum tissue of patients undergoing liposuction procedures for morbid obesity. This study standardized a closed system protocol which can be utilized for clinical grade stem cell derivation. Stages of cell growth and characterization of human adipose tissue-derived mesenchymal stem cells were also assessed along with the chromosomal stability in these in vitro cultures. Results Human adipose tissue-derived mesenchymal stem cells maintained their spindle-shaped morphology and were able to proliferate and renew, confirming their suitability for in vitro cultivation and generate clinical grade mesenchymal stem cells. Immunophenotyping indicates that the cells expressed cluster of differentiation (CD)73/CD90/CD105, mesenchymal stem-cell markers, while lacked CD34/CD45/ Human Leukocyte antigen-antigen D related (HLA-DR) expression (hematopoietic cell markers). A cell cycle study demonstrated growth kinetics under in vitro culture conditions. Human adipose tissue derived mesenchymal stem cells expressed normal cell karyotype by chromosomal G-banding indicating their genetic stability at Passage 5. Mesenchymal stem cells also demonstrated trilineage differentiation. Conclusions Availability of adipose tissue in abundance is a major advantage for clinical applications. Furthermore, detailed characterization of human adipose tissue-derived mesenchymal stem cells, their genomic stability and differentiation potential from stromal vascular fraction of human adipose tissue would help assist in tissue regeneration and repair.

Standardization and quality assessment for clinical grade mesenchymal stem cells from human adipose tissue.

BACKGROUND: Mesenchymal stem cells have immense potential in stem cell-based therapies, however there is a pre-requisite to develop a curative cell dose. Adipose tissue-derived mesenchymal stem cells are promising mainly due to their potential abundance, immunomodulatory effect and remarkable differentiation potential. Nevertheless, senescence may develop during their in vitro expansion due to the incidence of genetic instability. Hence, it is important to attain an ideal balance between mesenchymal stem cell growth, quality and genetic integrity before their clinical use. METHODS: Stromal vascular fraction was obtained from omentum tissue of patients undergoing liposuction procedures for morbid obesity. This study standardized a closed system protocol which can be utilized for clinical grade stem cell derivation. Stages of cell growth and characterization of human adipose tissue-derived mesenchymal stem cells were also assessed along with the chromosomal stability in these in vitro cultures. RESULTS: Human adipose tissue-derived mesenchymal stem cells maintained their spindle-shaped morphology and were able to proliferate and renew, confirming their suitability for in vitro cultivation and generate clinical grade mesenchymal stem cells. Immunophenotyping indicates that the cells expressed cluster of differentiation (CD)73/CD90/CD105, mesenchymal stem-cell markers, while lacked CD34/CD45/ Human Leukocyte antigen-antigen D related (HLA-DR) expression (hematopoietic cell markers). A cell cycle study demonstrated growth kinetics under in vitro culture conditions. Human adipose tissue derived mesenchymal stem cells expressed normal cell karyotype by chromosomal G-banding indicating their genetic stability at Passage 5. Mesenchymal stem cells also demonstrated trilineage differentiation. CONCLUSIONS: Availability of adipose tissue in abundance is a major advantage for clinical applications. Furthermore, detailed characterization of human adipose tissue-derived mesenchymal stem cells, their genomic stability and differentiation potential from stromal vascular fraction of human adipose tissue would help assist in tissue regeneration and repair.

Association of TNF-α, IL-10 and IL-6 promoter polymorphisms in pulmonary tuberculosis patients and their household contacts of younger age group.

Single nucleotide polymorphisms (SNPs) of cytokine genes have been found to be involved in the clinical outcome of Tuberculosis. The present study was aimed to identify the high risk genotypes in Tuberculosis patients and their household contacts. A total of 490 subjects were studied which includes 150 active pulmonary tuberculosis patients (APTB), 190 household contacts (HHC) and 150 healthy controls (HC). The SNPs of TNF-α (-308A/G), IL-10(-1082G/A) and IL-6(-174G/C) were performed by ARMs PCR. The IL-10 GA genotype showed significant association in APTB and HHC and was 2.3 times higher risk in APTB and 3.7 times in HHC compared to HCs. The A allele was found to be significantly associated with the risk of disease. The CC genotype of IL-6 was found to be significantly associated in APTB and an insignificant positive association in HHCs. The multifactor dimensionality reduction (MDR) analysis indicated that the genotypes of IL-6 were showing high risk with GA genotype of IL-10. In conclusion the gene interaction may be useful for identification of genotypes as biomarkers to distinguish high risk individuals.

In vitro allogeneic immune cell response to mesenchymal stromal cells derived from human adipose in patients with rheumatoid arthritis.

We investigated the regulatory activity of human adipose-derived mesenchymal stromal cells (MSCs) (n=10) towards immune cells in a cohort of 84 rheumatoid arthritis (RA) patients, 36 apparently healthy controls. We co-cultured MSCs with lymphocyte subsets of T, B, and T regulatory cells (Tregs). Levels of the pro- and anti-inflammatory markers (tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and interleukin-10 (IL-10)) were estimated in serum and co-culture supernatants. The study revealed a two-fold increase in the proportion of Tregs and an increased level of CD4+CD25+FoxP3. MSCs altered T cell, B cell, and Treg cytokine production during an anti-inflammatory immune response. The MSCs inhibited CD3+T cell-mediated TNF-α secretion, upregulated IL-10, and suppressed the production of autoantibodies against citrullinated protein antigens produced by B cells. These data offer insight into the interactions between allogeneic MSCs and immune cells, and elucidate the dose-dependent modulation of MSCs.

Cytokine production and mRNA expression in pulmonary tuberculosis patients and their household contacts of younger age group (15-25years).

Household contacts of tuberculosis patients are at high risk of infection and development of active disease. In this study we evaluated the cytokine production and mRNA expression of IFN-γ, TNF-α, IL-10&IL-6 stimulated with r32kDa M. bovis BCGAg in active pulmonary tuberculosis patients (APTB), household contacts (HHC) and healthy controls (HC). The results showed the stimulated levels of IFN-γ and TNF-α were low while IL-10 levels were high in APTB and HHC compared to HC. IL-6 has not shown any significant difference. The mRNA expression of TNF- α was 8 fold high in HCs compared to APTB and HHC. The IL-6 expression was 2.2 fold &1 fold less in APTB and HHC compared to HCs. Multinomial logistic regression analysis indicated that the stimulated levels of IFN-γ & IL-6 and sex significantly predicted the HHC group from HCs at p<0.05.In conclusion further follow up studies with r32kd antigen might help to identify the high risk individuals.

Evaluation of TNF-α, IL-10 and IL-6 Cytokine Production and Their Correlation with Genotype Variants amongst Tuberculosis Patients and Their Household Contacts.

BACKGROUND: Household contacts of diagnostically established tuberculosis (TB) patients are highly susceptible to disease development. It is surmised that cytokines perhaps play a synergistic and a prognostic role in the activation of the otherwise latent infection in these house hold contacts. Evaluation of the cytokines and any of their inherent polymorphisms might provide a useful diagnostic tool in evaluating the immune regulation and the progression of the disease. The cytokines thus released in a paracrine manner in serum may also provide an indirect measure of the cytokine function. OBJECTIVE: The present study was aimed to evaluate the levels of TNF-α, IL-10 & IL-6 cytokines and their correlation with genotype variants amongst tuberculosis patients and their household contacts. METHODS: The cytokine levels were estimated in serum by enzyme-linked immunosorbent assay (ELISA) and their polymorphisms were studied by amplification refractory mutation system polymerase chain reaction (ARMs PCR) in active pulmonary tuberculosis patients (APTB = 150), household contacts (HHC = 190), and healthy controls (HC = 150). RESULTS: The median values of TNF-α cytokine were significantly high among APTB and HHC compared to HCs (P< 0.0001 and 0.0001). IL-6 levels also were elevated among APTB compared to HHC and HC, and a significant difference was observed between APTB and HHC at P<0.0001; APTB & HC at P< 0.04; HHC & HC at P< 0.01. The IL-10 levels were low in APTB compared to HHC and HCs and no significant difference was observed. TNF-α/IL-10 ratio was significant and indicated Th1 predominance in APTB and HHC. IL-6/IL-10 showed pronounced Th1 expression in APTB and Th2 in HHC and HC. The ROC analysis indicated that both IL-10 and IL-6 can be used to decide the risk of exposed individual to a disease. The results of multivariate analysis indicate that IL-10 (-1082) GA genotype was significantly associated with p<0.028 in APTB. No significant association was observed between genotypes, other serum cytokine levels and clinical characteristics between APTB, HHC and HCs. CONCLUSION: Large sample size with follow-up at different time points may further illuminate the role of IL-10 and IL-6 cytokines as a prognostic marker in house hold contacts.

Mesenchymal Stromal Cell Therapy in MDR/XDR Tuberculosis: A Concise Review.

Multi-drug-resistant (MDR) tuberculosis is a major public health problem worldwide. Drug resistance arises due to non-compliance of antibiotic therapy. Herein, we explored the therapeutic options available ranging from conservative treatment approaches to alternate adjunct therapies such as mesenchymal stromal cell (MSC) therapy interventions. It is attractive to understand the scientific rationale of using cells as drugs, in particular mesenchymal stem/stromal cells. The review dwells and attempts to analyze the mechanistic approaches of the current treatment modalities to modern therapies. MSCs have demonstrated profound capacity to regenerate and repair. They appear to modulate that the activities of dendritic cells regulate T cells, both in vivo and in vitro. While there seems to be some benefit of such therapies, its use warrants further research. The merits and de-merits of autologous therapy/allogeneic therapy are ill understood. The challenges of requirement of large number of cells for infusion, the route of administration, choice of timing are complex issues that need to be addressed. Furthermore, the host immune responses, environmental factors and epigenetic mechanisms compound the problem. Although, clinical studies are being performed using autologous MSCs in different inflammatory models, it is important that such an intervention should be based on sound scientific rationale. The current review examines the immunomodulatory properties of MSCs, its interactions with other cell types, in assessing the basis for autologous/allogeneic cell-based therapies in the treatment of XDR/MDR tuberculosis.

Proliferation and differentiation potential of human adipose-derived stem cells grown on chitosan hydrogel.

Applied tissue engineering in regenerative medicine warrants our enhanced understanding of the biomaterials and its function. The aim of this study was to evaluate the proliferation and differentiation potential of human adipose-derived stem cells (hADSCs) grown on chitosan hydrogel. The stability of this hydrogel is pH-dependent and its swelling property is pivotal in providing a favorable matrix for cell growth. The study utilized an economical method of cross linking the chitosan with 0.5% glutaraldehyde. Following the isolation of hADSCs from omentum tissue, these cells were cultured and characterized on chitosan hydrogel. Subsequent assays that were performed included JC-1 staining for the mitochondrial integrity as a surrogate marker for viability, cell proliferation and growth kinetics by MTT assay, lineage specific differentiation under two-dimensional culture conditions. Confocal imaging, scanning electron microscopy (SEM), and flow cytometry were used to evaluate these assays. The study revealed that chitosan hydrogel promotes cell proliferation coupled with > 90% cell viability. Cytotoxicity assays demonstrated safety profile. Furthermore, glutaraldehyde cross linked chitosan showed < 5% cytotoxicity, thus serving as a scaffold and facilitating the expansion and differentiation of hADSCs across endoderm, ectoderm and mesoderm lineages. Additional functionalities can be added to this hydrogel, particularly those that regulate stem cell fate.

Fluorescent magnetic iron oxide nanoparticles for cardiac precursor cell selection from stromal vascular fraction and optimization for magnetic resonance imaging.

Fluorescent magnetic iron oxide nanoparticles have been used to label cells for imaging as well as for therapeutic purposes. The purpose of this study was to modify the approach to develop a nanoprobe for cell selection and imaging with a direct therapeutic translational focus. The approach involves physical coincubation and adsorption of superparamagnetic iron oxide nanoparticle-polyethylene glycol (SPION-PEG) complexes with a monoclonal antibody (mAb) or a set of antibodies. Flow cytometry, confocal laser scanning microscopy, transmission electron microscopy, iron staining, and magnetic resonance imaging were used to assess cell viability, function, and labeling efficiency. This process has been validated by selecting adipose tissue-derived cardiac progenitor cells from the stromal vascular fraction using signal regulatory protein alpha (SIRPA)/kinase domain receptor (KDR) mAbs. These markers were chosen because of their sustained expression during cardiomyocyte differentiation. Sorting of cells positive for SIRPA and KDR allowed the enrichment of cardiac progenitors with 90% troponin-I positivity in differentiation cultures. SPION labeled cardiac progenitor cells (1×10(5) cells) was mixed with gel and used for 3T magnetic resonance imaging at a concentration, as low as 12.5 µg of iron. The toxicity assays, at cellular and molecular levels, did not show any detrimental effects of SPION. Our study has the potential to achieve moderate to high specific cell selection for the dual purpose of imaging and therapy.

Evaluation of the autologous bone marrow mononuclear therapy and functional restoration in the scarred myocardium by imaging analysis.

A 62-year-old male patient with previous history of myocardial infarction, akinetic myocardial segments, and an ejection fraction of 31% with the NYHA class III category was selected for the autologous bone marrow (ABM)-derived mononuclear cell fraction injection during CABG surgery. Nitrate augmented myocardial tracer uptake was imaged by ECG gated SPECT pre- and 1 year post-ABM therapy, using radiotracer Tc99m Sestamibi. The baseline gated SPECT demonstrated full thickness infarct in 40% area of LAD territory. Bone marrow aspirate of 20.0 ml from sternum yielding a mono nuclear cell fraction of 4.5 × 10(7) cells/ml was suspended in 2.0 ml of sterile normal saline to be injected at eight sites of the injured myocardium. There were no apparent side effects due to the procedure, i.e., life threatening events, major bleeds, reaction, or shock. The case was followed at the end of 1, 3, 6 months by ECG and Holter monitor and ECG gated SPECT at the end of 12 months. The gated SPECT images demonstrated mild but definitely improved tracer uptake within part of the infarcted segments along with improvement in ejection fraction to 45%, and a clinical change in the NYHA Class to II. Cell-based therapy may offer benefits of induction of normal tissue microenvironment.

Impact of ethnicity, donor status and HLA matching on renal allograft survival: a single center study.

The role of histocompatibility testing in renal transplantation is passing through an immense debate on its utility in predicting long-term graft survival. The current study, which includes fifty-one patients with end-stage renal disease, aims at evaluating the impact of the HLA matching in live related donor (LRD) (parents, siblings and near relatives) and live unrelated donor (LURD) transplants on one year graft survival rates, in a single center. Patients were followed-up for one-year after renal transplantation and observed for renal complications inclu-ding infections and rejection. The incidence of acute rejection episodes was found to be lower in LRD transplantation complying with many reports published so far. HLA matching was found to be beneficial in obtaining better graft function and one-year graft survival rate. The current study found that patients from Far East of India have lower graft survival rates as against patients from other regions of the country. India, with its vast racial distribution, has a need to look into the ethnic variation and its impact on allograft survival.

Histocompatibility testing of cultivated human bone marrow stromal cells - a promising step towards pre-clinical screening for allogeneic stem cell therapy.

Mesenchymal stem cells (MSCs) lack major histocompatibility complex (MHC)-II and only show minimal MHC-I expression. Despite MSCs demonstrating T-cell anergy, there are no established methods to evaluate their suitability. It is crucial to evaluate the complete mismatch of MHC compatibility in view of the hypo-immunogenic nature and immunomodulatory properties of MSCs with respect to their proliferation potential (PP) and utility in terms of passage number. With bone marrow (BM) being the major source of MSCs, the use of these cells becomes even more complicated, due to many other receptors coming to fore and triggering alternative pathways. This prospective study included five BM aspirates for MSC cultures and five allogeneic peripheral blood mono nuclear cells (PBMNCs) from healthy volunteers. MHC compatibility was assessed by polymerase chain reaction-sequence specific primer (PCR-SSP). The PP and a T-cell response to MSCs was addressed in mixed cultures and evaluated on the basis of their stimulation index (SI). Allogeneic circulatory antibodies against the donor MSCs was performed by cytotoxicity assay. The PP of MSCs during interactions with PBMNCs (T-cells) demonstrated T-cell anergy and the response to circulatory antibodies was minimal, in consonance with other published reports. Although, the results are encouraging for potential clinical application of MSC transplantation, autologous is always preferable to allogeneic, at least until the long-term safety of these cells is established in clinical trials.

Rh antigen expression during erythropoeisis: Comparison of cord and adult derived CD34 cells.

OBJECTIVES: Concentrations of O(2) and CO(2) in the fetal circulation differ to that in maternal blood. Previous studies done in algae demonstrate the functional role of Rh antigen as CO(2) transporter. As a preliminary study, it was the aim of this project to compare the expression of Rh polypeptides on cord and adult red blood cell progenitors during ex vivo proliferation and differentiation of CD34(+) cells during erythropoeisis. MATERIALS AND METHODS: CD34 positive hematopoeitic progenitor cells were isolated from umbilical cord blood and adult peripheral blood using an immunomagnetic system and cultured in serum free medium containing erythropoietin in order to compel them along the erythroid lineage. Cultured cells were analyzed for cell surface marker expression by flow cytometry, using monoclonal antibodies to RhAG, Glycophorin A, Rh polypeptides, CD47 and Band 3. Cytospin analysis was also done to study the morphology of cultured cells. RESULTS: The appearance of cell surface markers analyzed on different days of culture varied slightly between samples. There was no evidence to suggest that RhAG, GPA, CD47 and Band 3 expression was any different between adult and cord derived cells. Nevertheless, the results of Rh antigenic expression suggest a reasonable difference between the two groups with adult sample derived cells showing higher and earlier expression than cord blood derived cells. These preliminary findings require further investigation. CONCLUSION: Comparing the expression of cell surface markers especially Rh polypeptides between adult and cord blood derived erythroid progenitors might assist in discerning their functions and could be valuable in the study of erythropoeisis.