Comparison of guidelines for biological ancillary materials used for the manufacture of gene and cellular therapy products in Asia.

BACKGROUND AIMS: Although biologiocal ancillay materials (AMs) have specific risk associated with their derivations, it plays key role to manufature cell and gene therapy (CGT) products. It is important to understand the regulation relevant to AMs for developers. METHODS: The authors investigated the guidelines and pharmacopeia (hereinafter referred to as "guidelines") for biological AMs used for the manufacture of CGT products in Asia (China, India, Japan, Korea and Taiwan). In addition, the authors benchmarked the relevant guidelines in the United States (US) and European Union (EU). RESULTS AND DISCUSSIONS: The guidelines could be classified into two types based on whether specific AMs are scoped: (i) general guidelines for risk assessment of AMs and (ii) guidelines for specific AMs. The authors compared the risk categories for each type of AM provided in the general guidelines between the US and China and the specific requirements for bovine serum and trypsin in the guidelines of China, Japan, Taiwan, US and EU. The authors further compiled in-depth descriptions of the respective regulations in China, India, Japan, Korea and Taiwan. There is limited availability of some guidelines for specific AMs. Moreover, there are no common requirements established across the surveyed countries and regions. Therefore, the authors suggest a risk assessment approach for AMs with consideration of their biological origin and traceability, production steps applied and ability to control or remove AMs from the final medicinal product over the CGT manufacturing process.

Nonclinical and quality assessment of cell therapy products: Report on the 4th Asia Partnership Conference of Regenerative Medicine, April 15, 2021.

The 4th Asia Partnership Conference of Regenerative Medicine (APACRM) was held online on April 15, 2021, to promote regulatory harmonization of regenerative medicine products throughout Asia. Recognizing domestic regulatory guidelines within each country and region, and their underpinning rationales, is an important initial step toward a convergence of regulations. The 4th APACRM consisted of an open dialog with regulatory agencies regarding nonclinical and quality settings for cell therapy products (CTPs) through industry presentations and panel discussions with regulatory agencies. The latest updates on regenerative medicine fields in each country and region, and specific regulatory schematics in Japan, were also introduced. The objective of this paper is to summarize the proceedings of the 4th APACRM for public dissemination and to foster further discussion in the future.

IL-17A Blockade Attenuates Obliterative Bronchiolitis and IFN-γ Cellular Immune Response in Lung Allografts.

Obliterative bronchiolitis (OB), characterized by fibrous obliteration of the small airways, is a major impediment to long-term survival in lung allograft recipients. We found previously that IL-17A is produced primarily by CD4+ T cells and γδ T cells after lung transplant in a mouse model of orthotopic lung transplant. The absence of either subset of T cells was compensated for by expansion of the other subset, which suggested that systemic blockade of IL-17A was necessary. To determine the specific role of IL-17A in the development of OB, we treated lung allograft recipients with an IL-17A antagonistic antibody. After IL-17A blockade, the incidence of OB was significantly reduced in lung allografts. IL-17A blockade also significantly attenuated the severity of acute rejection and overall lung fibrosis. The decreased OB incidence was associated with reduced lymphocyte recruitment, particularly CD8+ T cells and other IFN-γ-producing lymphocytes, to the lung allograft. Interestingly, IL-17A blockade led to an increase in the frequency of IL-17A-producing T-helper cell type 17 cells and γδ T cells in lung allografts, suggesting that IL-17A is a negative regulator of these T cells. Our data suggest that blocking IL-17A after lung transplant reduces the overall IFN-γ-mediated lymphocyte response and decreases the development of OB.

Toxicophore exploration as a screening technology for drug design and discovery: techniques, scope and limitations.

Toxicity is a common drawback of newly designed chemotherapeutic agents. With the exception of pharmacophore-induced toxicity (lack of selectivity at higher concentrations of a drug), the toxicity due to chemotherapeutic agents is based on the toxicophore moiety present in the drug. To date, methodologies implemented to determine toxicophores may be broadly classified into biological, bioanalytical and computational approaches. The biological approach involves analysis of bioactivated metabolites, whereas the computational approach involves a QSAR-based method, mapping techniques, an inverse docking technique and a few toxicophore identification/estimation tools. Being one of the major steps in drug discovery process, toxicophore identification has proven to be an essential screening step in drug design and development. The paper is first of its kind, attempting to cover and compare different methodologies employed in predicting and determining toxicophores with an emphasis on their scope and limitations. Such information may prove vital in the appropriate selection of methodology and can be used as screening technology by researchers to discover the toxicophoric potentials of their designed and synthesized moieties. Additionally, it can be utilized in the manipulation of molecules containing toxicophores in such a manner that their toxicities might be eliminated or removed.

Preclinical safety & toxicity evaluation of pooled, allogeneic human bone marrow-derived mesenchymal stromal cells.

BACKGROUND & OBJECTIVES: Administration of ex vivo-expanded human bone marrow-derived mesenchymal stromal cells (hBMMSC) obtained from single donors has shown therapeutic benefits in both preclinical and clinical studies. In this study, the safety, toxicity and biodistribution profiles of a pooled hBMMSC population, produced from three healthy donors were assessed in rodent and non-rodents. METHODS: The pooled hBMMSC population was characterized by their expression of various cell surface markers, differentiation potential and immunomodulatory activity. To establish in vivo safety of the pooled cells, these were administered by various injection routes into rodents and non-rodents to determine overall toxicity, biodistribution and tumorigenic potential in a series of preclinical studies. RESULTS: Single injections of hBMMSC at various doses through intravenous or intramuscular routes did not cause toxicity in rats and rabbits. In addition, repeat administration of hBMMSC was also well tolerated by rats, and no prenatal toxicity was observed by multiple administration in the same animal species. Ex vivo-expanded and cryopreserved hBMMSCs did not induce tumour formation in severe combined immunodeficient (SCID) mice. INTERPRETATION & CONCLUSIONS: Our results showed that the pooled hBMMSC population was non-toxic, non-teratogenic and non-tumorigenic in animals. Further studies need to be done to find out if it can be safely administered in human patients.

CD4 T Cells but Not Th17 Cells Are Required for Mouse Lung Transplant Obliterative Bronchiolitis.

Lung transplant survival is limited by obliterative bronchiolitis (OB), but the mechanisms of OB development are unknown. Previous studies in a mouse model of orthotopic lung transplantation suggested a requirement for IL-17. We have used this orthotopic mouse model to investigate the source of IL-17A and the requirement for T cells producing IL-17A. The major sources of IL-17A were CD4(+) T cells and γδ T cells. Depletion of CD4(+) T cells led to a significantly decreased frequency and number of IL-17A(+) lymphocytes and was sufficient to prevent acute rejection and OB. However, mice with STAT3-deficient T cells, which are unable to differentiate into Th17 cells, rejected lung allografts and developed OB similar to control mice. The frequency of IL-17A(+) cells was not decreased in mice with STAT3-deficient T cells due mainly to the presence of IL-17A(+) γδ T cells. Deficiency of γδ T cells also did not affect the development of airway fibrosis. Our data suggest that CD4(+) T cells are required for OB development and expansion of IL-17A responses in the lung, while Th17 and γδ T cells are not absolutely required and may compensate for each other.

Randomized, double-blind, phase I/II study of intravenous allogeneic mesenchymal stromal cells in acute myocardial infarction.

BACKGROUND AIMS: Cell therapy is promising as an exploratory cardiovascular therapy. We have recently developed an investigational new drug named Stempeucel (bone marrow-derived allogeneic mesenchymal stromal cells) for patients with acute myocardial infarction (AMI) with ST-segment elevation. A phase I/II randomized, double-blind, single-dose study was conducted to assess the safety and efficacy of intravenous administration of Stempeucel versus placebo (multiple electrolytes injection). METHODS: Twenty patients who had undergone percutaneous coronary intervention for AMI were randomly assigned (1:1) to receive intravenous Stempeucel or placebo and were followed for 2 years. RESULTS: The number of treatment-emergent adverse events observed were 18 and 21 in the Stempeucel and placebo groups, respectively. None of the adverse events were related to Stempeucel according to the investigators and independent data safety monitoring board. There was no serious adverse event in the Stempeucel group and there were three serious adverse events in the placebo group, of which one had a fatal outcome. Ejection fraction determined by use of echocardiography showed improvement in both Stempeucel (43.06% to 47.80%) and placebo (43.44% to 45.33%) groups at 6 months (P = 0.26). Perfusion scores measured by use of single-photon emission tomography and infarct volume measured by use of magnetic resonance imaging showed no significant differences between the two groups at 6 months. CONCLUSIONS: This study showed that Stempeucel was safe and well tolerated when administered intravenously in AMI patients 2 days after percutaneous coronary intervention. The optimal dose and route of administration needs further evaluation in larger clinical trials (http://clinicaltrials.gov/show/NCT00883727).

Does toll-like receptor-3 (TLR-3) have any role in Indian AMD phenotype?

Age-related macular degeneration (AMD) is a devastating disease that results in irreversible central vision loss. TLRs signaling pathway has been found to play an important role in AMD pathogenesis as evidenced by several studies. The objective of the study was to determine the single nucleotide polymorphism (SNP) changes in TLR3 in North Indian AMD patients. We recruited 176 patients comprising 115 AMD patients and 61 controls. Real time PCR was used to evaluate the SNP changes at rs3775291 locus. Pearson's χ(2) test was used evaluate association between various groups. No significant association in genotype and allele frequency was found in AMD patients as compared to control. The results suggest that AMD pathology in North Indian AMD patients is not affected by TLR3 signaling but it could be influenced by other genetic or environmental factors unique to North India.

In Vitro Drug Release Testing Method For Nepafenac Ophthalmic Suspension.

In vitro release test (IVRT) method is important to monitor batch-to-batch quality variations during pharmaceutical manufacturing and also to show the pharmaceutical equivalence of a generic product with the innovator. To fulfil regulatory requirements for approval of a generic ophthalmic suspension product, in vitro release study is required. No compendial or non-compendial method is available for IVRT of nepafenac ophthalmic suspension. Current research is aimed to screen various approaches using different conventional and non-conventional instruments to suggest the most suitable technique appropriate for nepafenac ophthalmic suspension followed by optimization of method parameters and validation. The trials used the paddle apparatus (USP Type-2) with dialysis sacs, the flow-through cell apparatus (USP Type-4), the rotating bottle apparatus, and the Franz diffusion cell apparatus. With the USP Type-4 apparatus drug release was found to be ∼ 83% in the simulated tear fluid (STF) of pH 7.4 in 120 min that increased to ∼ 97% upon the addition of surfactant sodium lauryl sulfate (SLS). With USP Type-2 and Franz diffusion cell apparatus, the drug release was either slow or not reaching close to the complete release. Whereas, in the case of the rotating bottle apparatus, a burst release profile was observed. The estimation of the drug release was done by the HPLC method and all the method validation parameters like specificity, accuracy, linearity, and precision were found to be within acceptance criteria.

A convolution-based in vitro-in vivo correlation model for methylphenidate hydrochloride delayed-release and extended-release capsule.

Delayed-release and extended-release methylphenidate hydrochloride (JORNAY PM®) is a novel capsule formulation of methylphenidate hydrochloride, used to treat attention deficit hyperactivity disorder in patients 6 years and older. In this paper, we develop a Level A in vitro-in vivo correlation (IVIVC) model for extended-release methylphenidate hydrochloride to support post-approval manufacturing changes by evaluating a point-to-point correlation between the fraction of drug dissolved in vitro and the fraction of drug absorbed in vivo. Dissolution data from an in vitro study of three different release formulations: fast, medium, and slow, and pharmacokinetic data from two in vivo studies were used to develop an IVIVC model using a convolution-based approach. The time-course of the drug concentration resulting from an arbitrary dose was considered as a function of the in vivo drug absorption and the disposition and elimination processes defined by the unit impulse response function using the convolution integral. An IVIVC was incorporated in the model due to the temporal difference seen in the scatterplots of the estimated fraction of drug absorbed in vivo and the fraction of drug dissolved in vitro and Levy plots. Finally, the IVIVC model was subjected to evaluation of internal predictability. This IVIVC model can be used to predict in vivo profiles for different in vitro profiles of extended-release methylphenidate hydrochloride.

Advantages of pooling of human bone marrow-derived mesenchymal stromal cells from different donors versus single-donor MSCs.

Mesenchymal stromal cells (MSC) from adult bone marrow are the most commonly used cells in clinical trials. MSCs from single donors are the preferred starting material but suffer from a major setback of being heterogeneous that results in unpredictable and inconsistent clinical outcomes. To overcome this, we developed a method of pooling MSCs from different donors and created cell banks to cater clinical needs. Initially, the master cell banks (MCBs) were created at passage 1 (P1) from the bone marrow MSCs isolated from of nine different donors. At this stage, MCBs from three different donors were mixed in equal proportion and expanded till P3 to create working cell banks. Further, the pooled cells and individual donor MSCs were expanded till P5 and cryopreserved and extensively characterised. There was a large heterogeneity among the individual donor MSCs in terms of growth kinetics (90% Coefficient of variation (CV) for cell yield and 44% CV for population doubling time at P5), immunosuppressive ability (30% CV at 1:1 and 300% CV at 1:10 ratio), and the angiogenic factor secretion potential (20% CV for VEGF and71% CV for SDF-1). Comparatively, the pooled cells have more stable profiles (60% CV for cell yield and 7% CV for population doubling time at P5) and exhibit better immunosuppressive ability (15% CV at 1:1 and 32% CV at 1:10 ratio ) and consistent secretion of angiogenic factors (16% CV for VEGF and 51% CV for SDF-1). Further pooling does not compromise the trilineage differentiation capacity or phenotypic marker expression of the MSCs. The senescence and in vitro tumourigenicity characteristics of the pooled cells are also similar to those of individual donor MSCs. We conclude that pooling of MSCs from three different donors reduces heterogeneity among individual donors and produces MSCs with a consistent secretion and higher immunosuppressive profile.

Regulatory systems and requirements for clinical trials of AAV-based gene therapies - Perspectives from six Asian countries or regions: Report from the 6th Asia Partnership Conference of Regenerative Medicine - April 20, 2023.

Gene therapies, which include viral-vector gene delivery, genome editing, and genetically modified cell therapy, are innovative treatments with the potential to address the underlying genetic causes of disorders and to provide life-changing value in terms of curing disease. Although adeno-associated virus (AAV)-based gene therapy is one of the most advanced types of gene therapy, far fewer AAV-based gene therapy studies have been conducted in Asia than in North America and Europe. The 6th Asia Partnership Conference of Regenerative Medicine (APACRM) was held on April 20, 2023 in Tokyo, Japan. APACRM Working Group 3 comprehensively analyzed the regulatory processes that occur prior to the initiation of clinical trials as well as the regulatory requirements for AAV-based gene therapies for six Asian countries or regions (China, India, Japan, Singapore, South Korea, and Taiwan). In this article, we report the outcomes of this conference, summarizing the regulatory requirements for initiating clinical trials for AAV-based gene therapies in terms of the laws, regulations, and guidelines for gene therapy; consultations or reviews required by the health authorities; points to consider for scientific reviews by the health authorities; and specific challenges to address when developing gene therapy products in these locations. Finally, we present several policy recommendations, including simplifying the regulatory review system for multiple scientific review areas; simplifying the regulatory consultation system; and providing training programs and regulatory guidance to support the advancement of gene therapy development in Asia.

Transplantation of islet-like cell clusters derived from human dental pulp stem cells restores normoglycemia in diabetic mice.

BACKGROUND AIMS: The success of islet transplantation for diabetes depends on the availability of an adequate number of allogeneic or autologous islets. Postnatal stem cells are now considered for the generation of physiologically competent, insulin-producing cells. Our group showed earlier that it is possible to generate functional islets from human dental pulp stem cells by using a serum-free cocktail in a three-step protocol. METHODS: We compared the yield of generated islet-like cell clusters (ICCs) from stem cells from pulps of human exfoliated deciduous teeth (SHED) and dental pulp stem cells from permanent teeth (DPSCs). ICCs derived from SHED were packed in immuno-isolatory biocompatible macro-capsules and transplanted into streptozotocin (STZ)-induced diabetic mice. Non-diabetic and diabetic controls were transplanted with macro-capsules with or without islets. RESULTS: SHED were superior to DPSCs. STZ diabetic mice alone and mice transplanted with empty macro-capsules exhibited hyperglycemia throughout the experiment, whereas mice transplanted with macro-capsules containing ICCs were restored to normoglycemia within 3-4 weeks, which persisted for >60 days. CONCLUSIONS: Our results demonstrate for the first time that ICCs derived from SHED reverse STZ diabetes in mice without immunosuppression and offer an autologous and non-controversial source of human tissue that could be used for stem cell therapy in diabetes.

Influence of hypoxia, high glucose, and low serum on the growth kinetics of mesenchymal stem cells from deciduous and permanent teeth.

The therapeutic potential of mesenchymal stromal cells depends on their ability to survive and proliferate under adverse in vivo scenarios in a particular disease. In most of the sites of injury, especially in diabetic wounds, there can be hypoxia, hyperglycemia, and ischemia, leading to a lack of nutrients. Hence, the aim of our present study was to investigate the influence of hypoxia, high glucose, and low serum concentrations on the growth kinetics and proliferative potential of human dental pulp stem cells from exfoliated deciduous teeth (SHED) and permanent teeth (DPSC). In this study we isolated two types of specialized stem cells from human dental pulp tissues, which were supposedly of neural crest origin, and cultured them in KO-DMEM medium supplemented with 10% fetal bovine serum (FBS). Both SHED and DPSC were characterized for standard CD surface markers, and their ability to differentiate into adipogenic and osteogenic lineages was tested. SHED and DPSC were exposed to either hypoxia or high glucose or low serum conditions, and their growth kinetics and differentiation potentials were compared with those of normal culture conditions. We found that SHED retained their phenotypic expression and differentiation potential under hypoxia, high-glucose, and low-serum conditions and exhibited a higher proliferation in terms of cell yield and a reduced doubling time compared to DPSC. Our findings clearly demonstrate for the first time that SHED are superior to DPSC as evidenced by their enhanced proliferation under adverse culture conditions.

Application of the continual reassessment method to dose-finding studies in regional anesthesia: an estimate of the ED95 dose for 0.5% bupivacaine for ultrasound-guided supraclavicular block.

BACKGROUND: Previously reported estimates of the ED95 doses for local anesthetics used in brachial plexus blocks vary. The authors used the continual reassessment method, already established in oncology trials, to determine the ED95 dose for 0.5% bupivacaine for the ultrasound-guided supraclavicular block. METHODS: A double-blind, prospective trial was scheduled for 40 patients of American Society of Anesthesiologists class I-III presenting for upper limb surgery and supraclavicular block. The study dose to be administered was arbitrarily divided into six dose levels (12, 15, 18, 21, 24, and 27 ml) with a priori probabilities of success of 0.5, 0.75, 0.90, 0.95, 0.98, and 0.99 respectively. A continual reassessment method statistical program created a dose-response curve, which would shift direction depending on the success or failure of the block. Our starting dose was 21 ml and the next allocated dose was reestimated by the program to be the dose level with the updated posterior response probability closest to 0.95. RESULTS: After recruitment of eight patients, our initial dose levels and associated probabilities were deemed too low to determine the ED95. Updated a prioris were calculated from the statistical program, and the study recommenced with a new starting dose of 30 ml. On completion, the ED95 dose was estimated to be 27 ml (95% CI, 24-28 ml). CONCLUSIONS: The continual reassessment method trial design provided a credible estimate for the ED95 dose for 0.5% bupivacaine for our technique of supraclavicular block and may be of value as a statistically robust method for dose-finding studies in anesthesiology.

Phenotypic and functional comparison of optimum culture conditions for upscaling of dental pulp stem cells.

Advances in dental pulp stem cell (DPSC) biology and behaviour have promised much in the field of regenerative medicine. Their recent use in clinical trials for bone repair enforces the notion that DPSCs can be used successfully in patients; however they display diverse characteristics under different culture conditions. Since the success of any stem cell culture is regulated by its own micro-environment, it is imperative to optimise the growth conditions and establish a generic protocol for maintenance and scale-up. This study focused on optimisation of long-term culture conditions of human exfoliated deciduous teeth (SHED) in comparison with DPSCs, employing three commonly used basal media - knockout Dulbecco's modified Eagle's medium (KO-DMEM), α-MEM and DMEM/F12. Based on their characterisation with respect to morphology, growth kinetics, cell surface marker expression, differentiation capacity and plating density, our findings suggest that cells can be expanded with the highest efficiency in KO-DMEM medium supplemented with 10% FBS. Additionally, under our standardised xeno-free (10% human plasma) growth conditions, DPSCs displayed and retained their multipotent attributes until late passages. The differences in the growth and differentiation characteristics between SHED and DPSCs are shown, and certify SHED can be a key element in tissue engineering.

Modulation of physical environment makes placental mesenchymal stromal cells suitable for therapy.

Low level of oxygen at the site of injury is likely to affect the viability and proliferation of the transplanted mesenchymal stromal cells (MSCs). Hence there is a need to understand the effect of the physical environment on transplanted stromal cells. Therefore, we have studied the effect of the duration of hypoxic exposure alone or in combination with normoxia on placenta derived mesenchymal stem cell (PDMSCs). PDMSCs and bone marrow MSCs (BMMSCs) were analysed under four different culture conditions, exposure to direct normoxia (N), direct hypoxia (H) and intermittent normoxia followed by hypoxia (NH) and intermittent hypoxia followed by normoxia (HN). The effect on morphology, proliferation, metabolic activity by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) and viability by 7AAD (7-amino-actinomycin D) were assayed, along with markers for MSCs and HLADR. No change in morphology, marker expression or HLADR was detected in N, H, NH or HN. An increase in proliferation rate, decrease in population doubling-time (PDT) and a relative increase in metabolic activity was strongly noted in the order: NH, N/HN and H. No significant difference was observed in the viability between N, H, NH or HN. A similar pattern was also observed in BMMSCS, indicating comparable suitability of PDMSCs in therapeutic applications. Thus we conclude that intermittent exposure to normoxia prior to hypoxic exposure is a better option than direct exposure to hypoxia. This may have clinical relevance in that they probably mirror the in vivo scenario of systemic delivery (NH) of cells as opposed to local delivery (H), thereby suggesting that systemic delivery is better than local delivery.

Intra-arterial allogeneic mesenchymal stem cells for critical limb ischemia are safe and efficacious: report of a phase I study.

BACKGROUND: Critical limb ischemia (CLI) caused by peripheral arterial disease is associated with significant morbidity and mortality. This condition is associated with a 30 % amputation rate as well as mortality levels which might be as high as 25 %. There is no pharmacological therapy available, but several reports have suggested that mesenchymal stem cells (MSCs) may be a useful therapeutic option. METHODS: This study, done at a university hospital, evaluated 13 patients for a phase I trial to investigate the safety and efficacy of intra-arterial MSCs in CLI patients. Eight patients with ten affected limbs were recruited for the study. As two patients (three limbs) died of ischemic cardiac events during the 6-month follow-up period, seven limbs were finally evaluated for the study. RESULTS: There was significant pain relief. Visual analog scale (VAS) scores decreased from 2.29 ± 0.29 to 0.5 ± 0.34 (p < 0.05), ankle brachial pressure index (ABPI) increased significantly from 0.56 ± 0.02 to 0.67 ± 0.021 (p < 0.01), and transcutaneous oxygen pressure (TcPO2) also increased significantly in the foot from 13.57 ± 3.63 to 38 ± 3.47. Similar improvement was seen in the leg as well as the thigh. There was 86 % limb salvage and six of seven ulcers showed complete or partial healing. CONCLUSION: It was concluded that intra-arterial MSCs could be safely administered to patients with CLI and was associated with significant therapeutic benefits.

Extracellular matrix protein mediated regulation of the osteoblast differentiation of bone marrow derived human mesenchymal stem cells.

The biomimetic approach of tissue engineering exploits the favorable properties of the extracellular matrix (ECM), to achieve better scaffold performance and tissue regeneration. ECM proteins regulate cell adhesion and differentiation through integrin mediated signal transduction. In the present study, we have examined the role of ECM proteins such as collagen type I, fibronectin, laminin and vitronectin in regulating the proliferation and osteogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs were grown on selected ECM protein treated tissue culture plates. The growth kinetics was assessed by calculating the doubling time of the cells on different ECM treated plates. The cells were directed to osteoblast lineage by growing them in osteogenic induction media for 21 day. Differentiation was evaluated at different time points by osteoblast differentiation associated gene expression, alkaline phosphatase (ALP) activity, histochemical staining for mineralized matrix and calcium quantification. The doubling time of hMSCs cultured on collagen type I was significantly low, which was followed by laminin and fibronectin treated plates. However, doubling time of hMSCs cultured on vitronectin treated plate was not significantly different than that of the untreated control. High ALP gene (ALPL) expression and associated enhancement of mineralization were observed on collagen type I, fibronectin and vitronectin treated plates. Collagen type I showed early onset of mineralization with high ALP activity and up-regulation of osteopontin, ALPL, bone sialoprotein and osteocalcin genes. Vitronectin also up-regulated these genes and showed the highest amount of calcium in the secreted mineral matrix. Therefore, we conclude that, ECM proteins indeed modified the growth patterns and induced the osteoblast differentiation of hMSCs. Our findings have significant implication for bone tissue engineering applications.