Significance of CD34 Negative Hematopoietic Stem Cells and CD34 Positive Mesenchymal Stem Cells - A Valuable Dimension to the Current Understanding.

BACKGROUND: The strategy to expand CD34+ hematopoietic stem cells (HSCs) is being increasingly practiced to meet the demand for a higher cell dose. This is for hematopoietic reconstitution in patients with higher body weights. Interestingly, literature reports show that CD34- (CD34 negative) cell population also possesses the potential to reconstitute the bone marrow & in a certain phase, converts them into CD34+ phenotype. The current practice of positive selection of CD34+ HSCs by eliminating rest of the (CD34-) population for expansion could probably pose a risk of losing valuable HSCs with good reconstitution potentials. MSCs (Mesenchymal Stem Cells) hold great promise for use in various regenerative medicine applications. International Society for Cellular Therapy (ISCT) defined MSCs to be CD34 negative; tissue resident MSCs and peripheral blood-derived MSCs express CD34 on their surface even in vitro, though up to limited passages. This interesting observation of CD34 positive expression displayed in vivo by non-hematopoietic cell types such as MSCs was intriguing, thus prompting a detailed review of its significance, if any. OBJECTIVE: Based on an extensive review, we strongly believe that CD34 expression in MSCs has some significant role in hematopoietic reconstitution & in regeneration of degenerated tissues. The concept of poor CD34 expression or stunted expression by certain MSCs should not be ignored. Several interesting research findings are in agreement with our assumption. However, it still leaves behind several unanswered questions that can only be addressed through detailed studies of phenotypic developmental pathways of MSCs.

Umbilical Cord Derived Mesenchymal Stem Cells Useful in Insulin Production - Another Opportunity in Cell Therapy.

BACKGROUND AND OBJECTIVES: Type 1 Diabetes Mellitus (T1DM) is an autoimmune disorder resulting out of T cell mediated destruction of pancreatic beta cells. Immunomodulatory properties of mesenchymal stem cells may help to regenerate beta cells and/or prevent further destruction of remnant, unaffected beta cells in diabetes. We have assessed the ability of umbilical cord derived MSCs (UCMSCs) to differentiate into functional islet cells in vitro. METHODS AND RESULTS: We have isolated UCMSCs and allowed sequential exposure of various inducing agents and growth factors. We characterized these cells for confirmation of the presence of islet cell markers and their functionality. The spindle shaped undifferentiated UCMSCs, change their morphology to become triangular in shape. These cells then come together to form the islet like structures which then grow in size and mature over time. These cells express pancreatic and duodenal homeobox -1 (PDX-1), neurogenin 3 (Ngn-3), glucose transporter 2 (Glut 2) and other pancreatic cell markers like glucagon, somatostatin and pancreatic polypeptide and lose expression of MSC markers like CD73 and CD105. They were functionally active as demonstrated by release of physiological insulin and C-peptide in response to elevated glucose concentrations. CONCLUSIONS: Pancreatic islet like cells with desired functionality can thus be obtained in reasonable numbers from undifferentiated UCMSCs in vitro. This could help in establishing a "very definitive source" of islet like cells for cell therapy. UCMSCs could thus be a game changer in treatment of diabetes.

Cryopreserved hepatic progenitor cells derived from human embryonic stem cells can arrest progression of liver fibrosis in rats.

Hepatocytes generated from human embryonic stem cells (hESCs) are considered to be an excellent candidate for restoring the liver function deficiencies. We have earlier standardized a three-step differentiation protocol to generate functional hepatocyte-like cells (HLCs) from hESCs, which expressed the major hepatic markers. We have also found that the HLCs remain stable and functional even after extended period of in vitro culture and cryopreservation. In the present study, we have aimed to investigate the therapeutic potential of cryopreserved-thawed hESC-derived hepatic progenitor cells following transplantation in carbon tetrachloride-induced fibrotic rat livers. Significant therapeutic effects, including improved hepatic histology and normal serum biochemistry of hepatic enzymes along with increased survival rate, were observed in the cell transplanted rats. This result is an encouraging indication to develop methods for clinical application of hESC-derived hepatic lineage cells.

Long-term culture and cryopreservation does not affect the stability and functionality of human embryonic stem cell-derived hepatocyte-like cells.

Human embryonic stem cells (hESCs) are predicted to be an unlimited source of hepatocytes which can pave the way for applications such as cell replacement therapies or as a model of human development or even to predict the hepatotoxicity of drug compounds. We have optimized a 23-d differentiation protocol to generate hepatocyte-like cells (HLCs) from hESCs, obtaining a relatively pure population which expresses the major hepatic markers and is functional and mature. The stability of the HLCs in terms of hepato-specific marker expression and functionality was found to be intact even after an extended period of in vitro culture and cryopreservation. The hESC-derived HLCs have shown the capability to display sensitivity and an alteration in the level of CYP enzyme upon drug induction. This illustrates the potential of such assays in predicting the hepatotoxicity of a drug compound leading to advancement of pharmacology.

Preparation and characterization of silver nanoparticle loaded amorphous hydrogel of carboxymethylcellulose for infected wounds.

There is a growing demand for an appropriate and safe antimicrobial dressing to treat infected deep wounds. An amorphous gel formulation (SNP-CMC), containing silver nanoparticles (SNPs) and carboxymethylcellulose (CMC), was prepared in one step by the reduction of silver nitrate in situ. Spectrophotometric and microscopic analysis revealed that the SNPs were 7-21 nm in diameter. In simulated wound experiments, SNP-CMC gel was found to absorb 80.48 ± 4.69% w/w of saline and donate 17.43 ± 0.76% w/w of moisture within 24h indicating its dual fluid affinity. Cytocompatibility of the gel was assessed by proliferation studies with primary human skin cells. The antimicrobial activity studies showed that SNP-CMC containing 50 ppm of SNPs was effective against the growth of both Gram negative and Gram positive strains including methicillin-resistant Staphylococcus aureus (MRSA). These results indicate that SNP-CMC could be ideal for the treatment of deep infected wounds.

Potential of Mesenchymal Stem Cell based application in Cancer.

Stem cell based treatments are being increasingly explored for their possible potential to treat various cancers. Mesenchymal stem cells believed to possess anti-tumor potential and are preferred for their properties like immune privileged nature, ability to migrate to the site of tumor and capability for multilineage differentiation. This tumor tropism property of MSCs could be utilized to deliver anti-tumor biological agents to the site of tumor. In a tumor micro-environment, MSCs are believed to play both, a pro-tumorigenic and an anti-tumorigenic role. However, this is dependent on a host of factors like, types of MSCs, its source, type of cancer cell line under investigation, in vivo or in vitro conditions, factors secreted by MSCs and interactions between MSCs, host's immune cells and cancer cells. Among several cytokines secreted by MSCs, TRAIL (Tumor necrosis factor related apoptosis inducing ligand) is reported to be pro-apoptotic for tumor cells. The MSCs from bone marrow and adipose tissue have been studied quite extensively. Deriving MSCs from sources such as umbilical cord blood and umbilical cord tissue is relatively easier. Umbilical cord tissue preferred for MSC derivation due to their abundant availability. These MSCs believed to up regulate TRAIL expression in MSC-cancer cell co-culture system resulting in induction of apoptosis in cancer cells. However, umbilical cord tissue derived MSCs needs to be studied for expression pattern of TRAIL in a co-culture system. We present a review article on different studies reporting both, pro-tumorigenic and anti-tumorigenic properties of MSCs.

Natural killer cells: In health and disease.

Natural killer (NK) cells constitute our bodies' frontline defense system, guarding against tumors and launching attacks against infections. The activities of NK cells are regulated by the interaction of various receptors expressed on their surfaces with cell surface ligands. While the role of NK cells in controlling tumor activity is relatively clear, the fact that they are also linked to various other disease conditions is now being highlighted. Here, we present an overview of the role of NK cells during normal body state as well as under diseased state. We discuss the possible utilization of these powerful cells as immunotherapeutic agents in combating diseases such as asthma, autoimmune diseases, and HIV-AIDS. This review also outlines current challenges in NK cell therapy.

Paracrine factors secreted by umbilical cord-derived mesenchymal stem cells induce angiogenesis in vitro by a VEGF-independent pathway.

Improvement in angiogenesis using mesenchymal stem cells (MSCs) is evolving as an option in patients with vascular insufficiencies. The paracrine factors secreted by MSCs have been attributed to the angiogenic response. This study was conducted to identify the factors secreted by umbilical cord-derived MSCs (UCMSCs) that might play a role in angiogenesis. To this aim, we evaluated the presence of well known proangiogenic factors in the conditioned media (CM) derived from UCMSCs by ELISA. While vascular endothelial growth factor (VEGF), a well known angiogenic factor, was not detected in the CM, gene expression was nevertheless detected in these cells. Further investigations revealed the presence of soluble VEGF receptors (sVEGF-R1 and R2) that were capable of neutralizing exogenous VEGF. Human umbilical cord vein-derived endothelial cells exposed in vitro to CM, in comparison to control media, showed improved migration (P<0.007) and capillary-like network formation (P<0.001) with no significant change in endothelial cell proliferation. The angiogenic response observed with the paracrine factors secreted by UCMSC could be due to the presence of significant levels of a metalloprotease and matrix metalloproteases-2 (237.4±47.1 ng/10(6) cells). Data suggest that a VEGF-independent pathway is involved in the angiogenic response observed with endothelial cells in the presence of UCMSC-CM.

A non-invasive method to evaluate the efficacy of human myoblast in botulinum-A toxin induced stress urinary incontinence model in rats.

PURPOSE: To develop a simple non-invasive method to assess the efficacy of a cell based therapy for treating stress urinary incontinence (SUI). MATERIALS AND METHODS: In this study, skeletal myoblasts were used as candidate therapy to reverse SUI. The SUI model was created in rats using periurethral injection of botulinum-A toxin injection. Two weeks later, the rats were administered saline and the level of continence in each botulinum-A toxin treated and control animals was assessed by the extent of voiding using metabolic cages. To determine the efficacy of myoblasts to reverse SUI, botulinum-A toxin treated incontinent rats were injected with either cultured human skeletal myoblasts or with buffered saline (sham control). Two weeks post implantation, the extent of continence was evaluated as mentioned above. RESULTS: The difference in void volume between botulinum-A toxin -treated and control rats were significant. Histological analysis of the urethra showed remarkable atrophy of the muscular layer. A significant reversal (P = .025) in the volume of voiding was observed in cell-implanted rats as compared to sham injected rats. Histological analysis of the urethra implanted with myoblasts showed recovery of the atrophied muscular layer in comparison to sham control. Immunofluorescence analysis of the cell injected tissues confirmed the presence of human myoblasts in the regenerated area. CONCLUSION: This simplified method of in vivo testing can serve as a tool to test the efficacy of new therapies for treating SUI.

Transplantation of Autologous Ex Vivo Expanded Human Conjunctival Epithelial Cells for Treatment of Pterygia: A Prospective Open-label Single Arm Multicentric Clinical Trial.

PURPOSE: To establish the efficacy and safety of ex vivo cultured autologous human conjunctival epithelial cell (hCjEC) transplantation for treatment of pterygia. METHODS: Twenty-five patients with pterygia were recruited at different centers across the country. Autologous hCjEC grafts were prepared from conjunctival biopsy specimens excised from the healthy eye and cultured ex vivo on human amniotic membrane mounted on inserts using a unique mounting device. The hCjEC grafts were then transported in an in-house designed transport container for transplantation. Post-surgery, the patients were followed up on days 1, 7, 14, 30, 90, and 180 as per the approved study protocol. Clinical outcomes were assessed by slit lamp examination, visual acuity, imprint cytology, fluorescein/rose bengal staining, Schirmer's test, and photographic evaluation three and 6 months post-transplantation. RESULTS: Two patients were lost to follow-up and final analysis included 23 cases. No recurrence of pterygium was observed in 18 (78.3%) patients; all of these eyes showed a smooth conjunctival surface without epithelial defects. Recurrence was observed in 5 (21.7%) patients at 3 months post-treatment. No conjunctival inflammation, secondary infections or other complications were reported. Adequate goblet cells were present in 19 (82.6%) patients at the site of transplantation. CONCLUSION: We have, for the 1(st) time, standardized a protocol for preparing autologous hCjEC grafts that can be safely transported to multiple centers across the country for transplantation. The clinical outcome was satisfactory for treating pterygia.

Status of stem cell based clinical trials in the treatment for diabetes.

Rapidly increasing number of diabetic patients across the world is a great challenge to the current therapeutic approach. Although the traditional method of rendering exogenous insulin is an established method of treatment, it is not sufficient and often causes lethal hypoglycemia. There is also a good amount of success with whole organ transplantation or Islet cells' transplantation. But this technique is limited with regards the availability of donors. Currently, many clinicians and researchers are involved in clinical studies using various different stem cells from embryonic as well as adult sources for the treatment of diabetes. In this review we have tried to discuss the results of various clinical trials using stem cells. We have also tried to look at various stem cell types and the routes of injections that are currently being followed world wide.

Dopaminergic cells, derived from a high efficiency differentiation protocol from umbilical cord derived mesenchymal stem cells, alleviate symptoms in a Parkinson's disease rodent model.

Mesenchymal stem cells (MSCs) could be an alternative to foetal cells in the treatment of several neurodegenerative diseases, especially Parkinson's disease (PD). We have previously demonstrated the functional efficacy of the undifferentiated bone marrow MSCs (BMMSCs) cultured in a xenofree conditions in PD animal models. We now demonstrate isolation of MSCs from the umbilical cord matrix tissue and assess their safety and efficacy to improve Parkinsonian symptoms in an in vivo animal model. The efficacy of MSCs from BM and umbilical cord in the PD animal mode has also been studied, and more importantly the efficacy of using differentiated UCMSC (D-UCMSCs) to dopaminergic phenotype. Phenotypically, UCMSCs expressed higher levels of SSEA4 compared to BMMSCs. Analysis of differentiated cells showed that D-UCMSCs expressed significant levels of Tyrosine Hydroxyalse and Nurr1 compared to D-BMMSCs. The in vivo efficacy of the differentiated and undifferentiated cell types in the Parkinsonian rats showed that D-UCMSCs improved the symptoms throughout a year of study. Differentiated cell types are potentially better for clinical use than the undifferentiated type, provided they are made available at the site of action in adequate numbers. MSCs are less immunogenic and immunomodulatory, which opens up the further possibility of using these cells in allogeneic settings. This could be a novel cell therapy application, especially when getting autochthonous cells is difficult.

In vitro and in vivo evaluation of (L)-lactide/ε-caprolactone copolymer scaffold to support myoblast growth and differentiation.

Skeletal muscle regeneration involves the activation of satellite cells to myoblasts, followed by their proliferation and fusion to form multinucleated myotubes and myofibers. The potential of in vitro proliferated myoblasts to treat various diseases and tissue defects can be exploited using tissue-engineering principles. With an aim to develop a biocompatible and biodegradable scaffold that supports myoblast growth and differentiation, we have developed a porous sponge with 70/30 L-lactide/ε-caprolactone copolymer (PLC) using a phase inversion combined with particulate leaching method. Degradation studies indicated that the sponge retained its structural integrity for 5 months in vitro and had undergone complete biodegradation within 9 months in vivo. The sponge supported human myoblasts attachment and its proliferation. Myoblasts seeded on the PLC sponge differentiated and fused in vitro to form myotubes expressing myosin heavy chain. Histological and molecular analyses of the PLC scaffolds seeded with green fluorescent protein-labeled human myoblasts and implanted ectopically under the skin in SCID mice demonstrated the presence of multinucleated myotubes expressing human muscle-specific markers. Our results suggest that PLC sponges loaded with myoblasts can be used for skeletal muscle engineering or for inducing muscle repair.

Establishment, characterization, and differentiation of a karyotypically normal human embryonic stem cell line from a trisomy-affected embryo.

Derivation of human embryonic stem cell (hESC) lines from chromosomally or genetically abnormal embryos obtained following preimplantation genetic diagnosis (PGD) is of immense interest to study various kinds of genetic disorders. In this study, we have established a new hESC line Relicell(®)hES4, isolated from an aneuploid embryo. Derivation of this cell line was achieved by isolation of the inner cell mass (ICM) by mechanical method. Karyotype analysis showed that the hESC line is euploid having 46 chromosomes, contrary to our expectations. The undifferentiated cells exhibited long-term proliferation capacity and expressed markers typical for hESC, such as OCT4, NANOG, and SSEA4. A comparative microarray study was carried out to analyze the transcription profile of Relicell(®)hES4 along with three other normal hESC line generated earlier in our lab. Relicell(®)hES4 manifested pluripotent differentiation potential both in vivo and in vitro. The cells were also induced to form neurons, cardiomyocytes, and pancreatic ß islets. The generation of a normal hESC line from an abnormal embryo points to the fact that even such embryos can be considered for deriving new hESC lines instead of discarding them. The data represented here are the first detailed report on characterization and differentiation of an Indian hESC line generated from a PGD analyzed embryo.

Defining Permissible Time Lapse between Umbilical Cord Tissue Collection and Commencement of Cell Isolation.

BACKGROUND: Umbilical cord tissue is a very rich source of mesenchymal stem cells. Instead of discarding this source we are banking the tissue along with cord blood for possible future cell based applications. The cord tissue needs to be transported and stored properly in order for it to be good enough for cell isolation at a later date. In this paper we have carried out a validation study to determine the maximum permissible time between cord tissue collection and beginning of cell culture process under defined conditions of temperature and collection media. METHODS: Ten cord tissue samples were used for this study. The umbilical cord tissue segments were transported and stored at 2 - 8°(C) for varying periods of time viz. 04, 11, 22 and 30 days in a defined medium after which MSCs were isolated and characterized by flow cytometry. Karyotypic studies were also performed on the isolated cells at the above time points. RESULTS: MSCs could be successfully isolated from 09 even samples after a storage period of 22 days and from 07 samples after a period of 30 days from the date of collection. There was no change in the morphology, immunophenotye, karyotypye and growth potential of the cells isolated from cord tissue after the maximum storage period of 30 days. CONCLUSION: The umbilical cord tissue is stable for as long as 22 days if stored at the recommended storage conditions of 2 - 8°(C) in the defined medium.

Radiolabeling of umbilical cord-derived mesenchymal stem cells for in vivo tracking.

Non-invasive methods for the assessment of distribution, homing, and retention of stem cells are desired for the successful demonstration of stem cell therapy. Cells labeled with (99m)Tc, (18)F, and (111)In have been reported for tracking the cells in vivo. However, they can be tracked only for a limited time due to the short half lives of these isotopes. In this context, stem cells labeled with (51)Cr would be appropriate for tracking cells for a longer period of time owing to their half life of 27.7 days. Here, we have isolated mesenchymal stem cells (MSCs) from umbilical cord tissue, characterized them, and attempted to radiolabel them with (51)Cr for mapping the fate of transplanted MSC cells after an intravenous injection via the tail vein in small animals.

Efficacy and safety of autologous cultured melanocytes delivered on poly (DL-lactic acid) film: a prospective, open-label, randomized, multicenter study.

BACKGROUND: Small vitiliginous patches have been treated with epidermal grafts or their cell suspensions. In an attempt to overcome some of the shortcomings of cell suspension delivery, we have delivered melanocytes on a polymeric film. OBJECTIVES: To evaluate the clinical effectiveness of a cultured graft consisting of autologous cultured melanocytes on a poly (DL-lactic acid) (PLA) film in subjects with stable vitiligo. METHODS: A prospective open-label, randomized, multicenter clinical trial was conducted with 22 patients. Each subject was treated with cultured graft and polyurethane dressing (control arm) after epidermal ablation and followed for up to 9 months. The extent of repigmentation in the treated sites was compared with that control sites at days 90, 180, and 270. RESULTS: In the treatment arm, a minimum of 70% repigmentation was observed in five subjects at day 90; nine at day 180, and 10 at day 270. In the control arm, only one subject showed repigmentation until day 270. None of the test sites reported any recurrence of vitiliginous patches by the end of the study. CONCLUSIONS: Cultured melanocytes delivered on PLA film were efficacious and safe when applied on patients with stable vitiligo.

Establishment of a mesenchymal stem cell bank.

Adult stem cells have generated great amount of interest amongst the scientific community for their potential therapeutic applications for unmet medical needs. We have demonstrated the plasticity of mesenchymal stem cells isolated from the umbilical cord matrix. Their immunological profile makes it even more interesting. We have demonstrated that the umbilical cord is an inexhaustible source of mesenchymal stem cells. Being a very rich source, instead of discarding this tissue, we worked on banking these cells for regenerative medicine application for future use. The present paper gives a detailed account of our experience in the establishment of a mesenchymal stem cell bank at our facility.

Molecular and cellular characterization of expanded and cryopreserved human limbal epithelial stem cells reveal unique immunological properties.

Transplantation of ex vivo expanded autologous limbal stem cells into the diseased eye of patients with limbal stem cell deficiency (LSCD) has been in practice worldwide. However, isolation of limbal tissue from the normal eye of the patient with unilateral LSCD still remains a major concern for the donor. More importantly, autologous cell transplantation is not a viable option for patients with bilateral LSCD. The objective of the current study was to determine the expansion potential of human limbal epithelial stem cells (hLESCs) for their possible use in allo-transplantation. A total of six limbal biopsy samples were cultured and expanded in vitro up to passage level 1 (P-1), at which point the hLESCs were cryopreserved. Semi-quantitative RT-PCR and immunophenotypic analysis revealed that hLESCs obtained before and after cryopreservation retained the expression of major limbal epithelial stem cell markers such as p63, SSEA-4, ABCG2, cytokeratin 19 (CK19), integrin ß1 and vimentin. One notable difference was that while P-0 hLESCs expressed HLA-DR mRNA, no HLA-DR gene expression was observed with the expanded and cryopreserved samples. Human LESCs did not express costimulatory proteins CD80 or B7-DC but expressed significant levels of CD86, B7-H1 and HLA-ABC molecules on the cell surface. Treatment of hLESCs with IFN-γ induced the expression of HLA-DR, indoleamine 2,3-dioxygenase (IDO) and HLA-G on these cells. Cultured hLESCs were unable to stimulate allogeneic T cell proliferation in vitro even in the presence of pro-inflammatory cytokine, IFN-γ. These results indicate that cryopreserved hLESCs are non-immunogenic in nature and express negative immunoregulatory molecules which may be critical for their survival in an allogeneic environment.

Tansplantation of autologous bone marrow derived mesenchymal stem cells trans-epicardially in patients undergoing coronary bypass surgery.

BACKGROUND: Myocardial Infarction (MI) hampers cardiac performance by ventricular remodeling which is a major cause of heart failure or death. Conventional drug therapies like beta blockers, angiotensin-converting enzyme may delay remodeling but there is no single therapeutic regimen available that can prevent or reverse the process of myocardial injury. Interventional therapies and surgical procedures improve or normalize coronary blood flow greatly. Experimental data suggests that bone marrow derived Mesenchymal Stem Cells (MSCs) may contribute to the healing of Myocardial infarction. We present our findings on the use of bone marrow derived MSCs for Myocardial Infarction wherein the cells were injected in and around the infarct region epicardially during coronary bypass surgery. METHODS & MATERIALS: 31 patients selected to undergo Coronary Artery Bypass Graft (CABG) as a treatment option for myocardial infarction formed the subject matter of our study. One patient withdrew consent before receiving our therapy and was excluded from the study. 15 patients (all men, average age 57) formed the test arm who underwent CABG plus Bone Marrow Mesenchymal Stem Cells (BMMSCs) transplantation whereas another 15 patients underwent conventional CABG only (14 men and 1 woman, mean age 57) served as the control arm. The cell transplantation consisted of injecting BMMSCs in the border zone of the clearly visible infarcted area transepicardially. The absolute number of MSCs injected ranged between 3 million and 26 million cells. RESULTS: The data for change from baseline in the area of infarct was collected at 3 months and 6 months during the study and analyzed using paired t-tests. The mean percentage perfusion improvement from baseline in the area of infarct supplied by the Left Anterior Descending Artery (LAD) was higher in the cases (35.8%) as compared to the controls (11.3%) at 3 months post treatment (p value < 0.05). There were three cases of arrhythmia, and none of the adverse events recorded were due to the investigational product. Improvement in the ejection fraction was similar in the cases and controls. CONCLUSIONS: This study demonstrates that trans-epicardial uses of mesenchymal stem cells are very safe and feasible. Correction of perfusion defect is very encouraging. Larger studies using higher doses of mesenchymal stem cells may bring about better understanding.