Open Access Short communication

Haploid Plant Regeneration from Unpollinated Ovules of Cucumis melo L. Var. Conomon cv. Mudicode

Sunita Premnath Koli, Hosakatte Niranjana Murthy

Biotechnology Journal International, Page 605-613
DOI: 10.9734/BBJ/2013/5417

Major objective of the present work was to induce haploid plants in Cucumis melo var. conomon cv. Mudicode (Common name: Kani vellari). Ovules of Kani vellari plants were collected one day before anthesis and were cultured in vitro on MS medium supplemented with IAA, IBA, NAA, 2, 4-D, KN, BAP, TDZ and 2-iP (1.0, 2.0 and 5.0 µM) and BAP/TDZ/KN (1.0, 2.0 and 5.0 µM) in combination with IAA, IBA, NAA, 2, 4-D (1.0, 2.0 and 5.0 µM). The cultured ovules involved in callus induction and an optimum quantity of 93.15 mg of callus from cultured ovules was obtained on the MS medium fortified with 1 µM BAP + 2 µM NAA. The callus induced on medium supplemented with 1 µM BAP+2 µM NAA was subcultured on MS medium supplemented with 5 µM BAP was involved in shoot organogenesis and developed a maximum of 5.55 shoots. The rooting of the regenerated shoots was achieved on MS medium supplemented with 1 µM IAA. Cytological analysis of the root tips of regenerated plants confirmed their haploid nature.

Open Access Original Research Article

Establishment of Agrobacterium-Mediated Transformation System in Sweet Potato (Ipomoea batatas) by Culture of Leaf Segments for Functional Analysis of ASG-1, an Apomixis-Specific Gene

Lanzhuang Chen, Chengti Xu, Zhaosheng Du, Takuro Hamaguchi, Toru Sugita, Hiroaki Ichikawa, Liming Guan

Biotechnology Journal International, Page 458-470
DOI: 10.9734/BBJ/2013/4582

Aims: In order to do the functional analysis of apomixis-specific gene (ASG-1), which was isolated from apomictic guineagrass, the sweet potato was used to establish an Agrobacterium-mediated transformation system.

Study Design: At first, plant regeneration was achieved from the culture of leaf segments of sweet potato. Based on it, a binary vector pSMA35H2-NG for transformation of ASG-1 was used for establishment of a suitable procedure for plant regeneration of transformants.

Place and Duration of Study: Faculty of Environmental and Horticultural Science, Minami Kyushu University, between June 2009 and December 2012.

Methodology: The leaf segments were used for somatic embryogenesis and plantlets regeneration. For the preliminary transformation, a GUS gene set in pSMA35H2-NG was introduced into the Agrobacterium strain GV3101/PMP9, and the Agrobacterium was used to infect the callus derived from leaf segments of sweet potato “Miyazakibeni” and the callus derived from seeds of rice “Nipponbare”. For the plasmid construction, the GUS was replaced by ASG-1, named as pSMA35H2/ASG1. The resultant plasmid was mobilized into Agrobacterium strain GV3101/PMP9 for transformation. For detection of ASG-1, DNAs of the transgenic plantlets were used for PCR, using the primers designed according to ASG-1 and hygromycin, respectively.

Results: 1) When the leaf segments were sterilized with sodium hypochlorite solution of 0.3% and 0.4% for 15 min, 100% of surviving rates was achieved. And the segments cultured on Murashige and Skoog (1962) gave 100% of callus formation rates. 2) When the calli were placed onto Komamine and Nomura (1998) medium for differentiation, somatic embryogenesis was obtained with white color and grain-like tissue, and plantlets with multiple shoot-like tissues were obtained from the somatic embryo. 3) For the preliminary transformation, the calli showed GUS blue spots gradually on the surface. 4) When the pSMA35H2/ASG1 was used to the transformation of the embryogenic calli, the plantlets were developed through multiple shoots. 5) The specific bands of ASG-1 and hygromycin were observed from the PCR products of the plantlets’ DNAs, respectively.

Conclusion: Overall the above results, the procedure using the binary vector pSMA35H2/ASG1 containing ASG-1 revealed, as the first case, that Agrobacterium-mediated transformation system in sweet potato was established using the culture of leaf segments in this study.

Open Access Original Research Article

Treatment of Wastewaters Generated by Surfactant-Enhanced Washing of Soils in an Aerobic Biofilter Inoculated with a Consortium of Hydrocarbon Degraders

M. Zacarías-Salinas, E. I. Garcia-Peña, L. G. Torres

Biotechnology Journal International, Page 471-484
DOI: 10.9734/BBJ/2013/4010

Aims: The aims of this work were 1) To evaluate the performance of a submerged biofiltration system for the treatment of a surfactant-enriched wastewater that had been generated by a soil washing process. 2) To evaluate the effect of the flux and organic load over the performance of the system. 3) To determine the microbial evolution as an effect of the flux at different lengths of the biofilter by using a denaturing gradient gel electrophoresis (DGGE) analysis.

Study Design: A three factorial design was used to evaluate the effect of different fluxes and organic loads over the performance of a continuously operated submerged aerobic biofilter. The DGGE technique was employed to determine microbial changes in the biofilter.

Place and Duration of Study: The study was carried out at the Bioprocess Laboratory, Bioprocesses Department UPIBI-IPN, Mexico. The experimental stage lasted approximately eight months and the DGGE analysis four months more.

Methodology: Contaminated soil was physicochemical and microbiologically characterized. A total of 70 kg of contaminated soil was washed using a 1:3 ratio soil/surfactant solution (0.5% Sulfopon 30-SP30). The surfactant-enriched wastewater was then treated in a submerged biofilter. The biofiltration system consisted of a column with a length of 50 cm and diameter of 12 cm. The biofilter was packed with tezontle with an average diameter of 0.2-0.4 cm and 70% void space. The biofilter working volume was 4.5 L. The samples of the packing material for the DGGE analysis were obtained from the ports located along the biofilter: at the wastewater inlet, at the middle of the column and at the outlet. After DNA extraction with a Power Soil DNA Isolation Kit (MO BIO), PCR (polymerase chain reaction) analysis was conducted. The 16S rRNA gene was amplified using universal bacterial primers. The data obtained by DGGE analysis for the microbial population developed in the biofilter were further analyzed by the Jaccard similarity coefficient.

Results: The soil contained 14,704 mg/kg TPH. BTEX compounds were not found, and only two different PAHs were found in the soil samples: benzo-fluoranthene and benzopyrene, at concentrations of 0.1280 and 0.0682 mg/kg of soil, respectively. During the surfactant-aided soil washing, the highest removal percentage of the oil removed from the soil was 59% with 0.5% SP30. The wastewater generated after the soil washing process contained, in average 1,329 mg COD/L and 211 mg/L of grease and oil. Higher COD removals were obtained at a flux of 0.4 L/h for both of the COD initial concentrations. While the highest removal was 78.27%, determined at an initial COD concentration of 300 mg/L. When applying fluxes of 0.28 and 0.40 L/h at a higher initial COD concentration, the COD removals were increased; this was not the case for a flux of 0.63 L/h. For a given initial COD concentration, the removal efficiencies were higher for lower fluxes. Analysis of the similarity between the microbial populations for varying fluxes and levels along the length of the biofilter was determined by the Jaccard (JI) index. The results showed that the initial microbial populations (t0) have low similarities with the developed microbial populations at the different conditions tested.

Conclusion: Both the flux and the initial COD concentration had an impact on COD removal and the microbial concentration in the column. The COD removal percentages were similar at fluxes of 0.28 and 0.63 L/h. The highest removal percentage of 78.27% was obtained at a flux of 0.4L/h; this finding was in agreement with the highest microbial count and the specialization of microbial populations (less diversity). In general, it was shown that the flux had an effect on changes in microbial population. Greater effects were observed on the microbial population due to the position along the reactor, e.g., the greatest differences were found at the different levels of the biofilter.

Open Access Original Research Article

Co-digestion of Food Waste and Human Excreta for Biogas Production

S. O. Dahunsi, U. S. Oranusi

Biotechnology Journal International, Page 485-499
DOI: 10.9734/BBJ/2013/4476

The ever increasing cost of fossil fuels and its attendant pollution menace has provided the pedigree to consider alternative sources of energy. An investigation was launched into the design and construction of an Anaerobic Digester system from locally available raw materials using local technology and the production of biogas from food wastes and Human excreta generated within a University campus. The experiment lasted for 60 days using a 40-liters laboratory scale anaerobic digester. The volume of gas generated from the mixture was 84,750cm3 and comprised of 58% CH4, 24% CO2, and 19% H2S and other impurities. The physico-chemistry of the feedstock in the digester revealed an initial drop in pH to more acidic range and a steady increase 4.52 – 6.10. The temperature remained relatively constant at mesophilic range: 22.0ºC– 30.5ºC throughout the study. The Carbon/Nitrogen (C/N) ratio of the feedstock before digestion was within 139:1. Population distributions of the microflora show aerobic and anaerobic bacteria to include Klebsiella spp, Bacillus spp, Escherichia coli, Clostridium spp and a methanogen of the genera Methanococcus. In most developing nations of Sub-Saharan Africa where biomass is abundant, and where biogas technology is in its infant stage, the anaerobic digestion system could be the much awaited solution.

Open Access Original Research Article

Kinetic Study of Neem Biodiesel Production

M. Elkadi, A. E. Pillay, J. Manuel, S. Stephen, M. Z. Khan

Biotechnology Journal International, Page 500-508
DOI: 10.9734/BBJ/2013/4434

Aims: The kinetic features of neem biodiesel production were studied to establish the conversion parameters that govern optimal product yield in minimum reaction span. The mechanistic features of the dual acid/base catalytic conversion were investigated to gain an insight into the influence of methoxide nucleophilic attack and catalytic restoration on reaction yield in the final stage.

Study Design: The reaction kinetics was investigated by UV-Vis absorption spectrophotometry. Neem biodiesel was extracted from neem oil via a two-stage chemical process. The neem oil feedstock is comparatively high in free fatty acids and its reduction is facilitated by an initial acid-catalysed pre-treatment. The second stage constitutes base-catalyzed transesterification to neem biodiesel and the glycerol by-product at 55ºC. The kinetic study focused on the second stage, which is underexplored with neem oil.

Place and Duration of Study: Chemistry Department, Arts and Sciences, The Petroleum Institute, Abu Dhabi, UAE.

Methodology: Suitable time intervals were selected to monitor the transesterification, and the absorbances of the resulting biodiesel were recorded in the far visible region at
700 nm (lmax.).

Results: It was found that the base-catalyzed reaction is rapid (<300 s) at the stipulated temperature and reaches completion after significant conversion to the biodiesel product. Absorbances were recorded after 1-minute cooling in an ice-water bath. Graphical delineation of the results revealed that the transesterification step conforms to zero-order kinetics. The difficulty encountered in making measurements was the fluctuating absorbances due to the separation of the phases – the rising biodiesel and the sinking glycerol.

Conclusion: The purpose of this study assists in defining the rate determining stage associated with the process. The biodiesel yield is 65% and inhibition of the reaction at lower temperatures or introduction of an inhibitor could be considered to prolong the final stage to acquire improved biodiesel yields.

Open Access Original Research Article

Optimization of Cellulase (E.C. 3.2.1: 4) Production Using Penicillium citrinum MTCC 9620 in Solid State Fermentation

G. Ghoshal, U. C. Banerjee, U. S. Shivhare

Biotechnology Journal International, Page 509-523
DOI: 10.9734/BBJ/2013/4135

Aim: The objective of the present work was to optimize the environmental parameters for Cellulase (1,4 β-endoglucanase, E.C.3.2.1:4) production using Penicillium citrinum MTCC 9620 in Solid State Fermentation.

Study Design: One unit of Cellulase (1,4 β-endoglucanase, E.C.3.2.1:4) activity is defined as the amount of enzyme producing 1μmole of glucose equivalent/min measured using UV visible spectrophotometer at 540 nm.

Place and Duration of Study: Food Technology laboratory of Dr. S. S. Bhatnagar University Institute of Chemical Engineering & Technology, Panjab University, Chandigarh between January and June 2011.

Methodology: Penicillium citrinum MTCC 9620 was maintained on potato dextrose agar (PDA) at 4°C. For Cellulase production Czapek Yeast Extract medium was used as moistening medium. Incubation temperature, pH, incubation time and other parameters like suitable substrate, pre-treatment of the substrate on production of Cellulase in Solid State Fermentation (SSF) was optimized using agricultural residues by Penicillium citrinum MTCC 9620. Microscopic and Spectral properties of substrates were determined to detect the structural changes after pre-treatment.

Results: Production of extracellular Cellulase was greatly affected by variation in substrates, pre-treatments of substrate and variation in pH and incubation temperature. Cellulase activity was significantly (p < 0.05) higher when alkali treated wheat bran was used as substrate than untreated substrate. Among three substrates and their three pre-treatment conditions, It has been observed that alkali treated wheat bran was the most suitable substrate for maximum cellulase production (12.56± 0.097U/mL) at pH 5.5 and 30ºC without any extraneous nitrogen source by Penicillium citrinum MTCC 9620 after 120 h of fermentation time. SEM study revealed that during alkali treatment the solid surface become rough which results growth of fungus eventually maximum cellulase production.

Conclusion: P. citrinum MTCC 9620 is one of the potential cellulase producing fungal strain. Optimum condition of cellulase (1,4 β-endoglucanase, E.C.3.2.1:4) production by P. citrinum MTCC 9620 was 30°C temperature, 5.5 pH when alkali treated wheat bran was used as substrate. Growth kinetics of P. citrinum MTCC 9620 was studied and it showed adequacy of fit to Monod Model to describe the growth pattern of P. citrinum MTCC 9620 in SSF at 30°C for 120 h incubation period.

Open Access Original Research Article

Dynamic Principle of Center of Mass in Human Walking

Yifang Fan, Yubo Fan, Zhiyu Li, Mushtaq Loan, Changsheng Lv

Biotechnology Journal International, Page 524-544
DOI: 10.9734/BBJ/2013/4724

Assuming the ground reaction force of both feet to be the same in the same phase of a stride cycle, we establish the relationships between the time of initial foot contact and the ground reaction force, acceleration, velocity, displacement and average kinetic energy of center of mass. We employ the dispersion to analyze the effect of the time of the initial foot contact that imposes upon these physical quantities. We present results of an analytic and numerical calculation that studies the relationship between the time of initial foot contact and the ground reaction force of human gait and explores the dynamic principle of center of mass. Our study reveals that when the time of one foot's initial contact falls right in the middle of the other foot's stride cycle, these physical quantities reach extrema. An action function has been identified as the dispersion of the physical quantities and optimized analysis used to prove the least-action principle in gait. In addition to being very significant to the research domains such as clinical diagnosis, biped robot's gait control, the exploration of this principle can simplify our understanding of the basic properties of gait.

Open Access Original Research Article

Structural Comparison of Wild and Mutated Matrix Protein of Nipah Virus via in-silico Approach

Sudhir kumar Pal

Biotechnology Journal International, Page 545-555
DOI: 10.9734/BBJ/2013/4051

Aims: To make three dimensional structure of native and mutated matrix protein (M) of Nipah Virus (NiV) and to establish conformational and functional comparison between the two.

Study Design:  All in-silico analysis were performed using various online and offline software.

Place and Duration of Study: Department of Biotechnology and Bioinformatics, Padmashree Dr. D.Y.Patil Vidyapeeth, Navi Mumbai, February 2013.

Methodology: The protein’s physicochemical properties were characterized and 3D model of both the normal and the mutated protein were created using Phyre2.   Single point mutation of S147G is recorded which leads to altered structure formation. Both the models were evaluated and compared conformationally.

Results: 4G1GB based structures were modeled by phyre2 and minimized energies recorded were-16760.041 kJ/mol for native and -16563.029 kJ/mol for the mutated protein. Structure validation proved that both the native and mutated structures were reliable. Formation of 3 H-bonds make mutated M structure slightly more stable than the native one.

Conclusion: NiV, one of deadliest pathogen, needs to be checked immediately. More information gain is needed by performing wet lab analysis. This work might help understand the functional difference between native and mutated M protein and can be used as the potent drug target via applying rational drug designing approach.

Open Access Original Research Article

Improved Sample Preparation for PCR-Based Assays in the Detection of Xanthomonads Causing Bacterial Leaf Spot of Tomato

E. R. Mbega, J. Adriko, C. N. Mortensen, E. G. Wulff, O. S. Lund, R. B. Mabagala

Biotechnology Journal International, Page 556-574
DOI: 10.9734/BBJ/2013/3810

Aims: To develop a sampling procedure for PCR-based screening of bacterial leaf spot (BLS)-causing xanthomonads without DNA extraction from infected tomato plants.

Place and Duration of Study: University of Copenhagen, Denmark and Sokoine University of Agriculture, Morogoro, Tanzania between July 2008 and November 2010.

Methodology: Flinders Technology Associates (FTA®) plant cards and Chromatography paper or Whatman® paper strips (WPS) were spotted with bacterial suspensions from 24-h-old cultures from reference strains of BLS-causing xanthomonads, or sap obtained by grinding or hand maceration of plant tissue, were used as templates in PCR reactions or isolation of live bacterial cells on Nutrient agar (NA) media. Samples were tested by PCR with Xan 7 genus/-specific Xanthomonas primers or in multiplex with 26S rRNA primers. Isolation of bacteria was done by streaking aliquots of 75 µl of a suspension from a disc (2-mm-punch by Harris Micro Punch®) in triplicate, removed from each of the FTA plant card and WPS onto NA media. 

Results: The FTA plant card spotted with pure cultures of reference strains of xanthomonads and sap from grinding or direct maceration of plant tissue resulted in more clear PCR bands (402 bp) and (594 bp of rRNA gene in multiplex) than the WPS samples. Sensitivity of detection by the FTA paper-based PCR was ≈ 5.0 x 102, while that of the WPS was > 1.0 x 103 CFU/ml. The WPS (but not the FTA) was proved to be useful for saving living bacteria cells for up to one week of storage at ambient temperatures.

Conclusion: Both FTA plant card and WPS can be used for PCR detection of BLS-causing xanthomonads in tomato. However, the FTA plant card is recommended as it produced clearer PCR products than WPS. WPS is recommended for experiments requiring isolation of live bacterial cells on NA media.

Open Access Original Research Article

Characterization of Dairy Effluents by Physicochemical Parameters

Vishakha Sukhadev Shivsharan, Minal Wani, M. B. Khetmalas

Biotechnology Journal International, Page 575-580
DOI: 10.9734/BBJ/2013/3640

Aims: The dairy industry faces growing scrutiny of its environmental stewardship.  The potential impact of an individual operation on the environment varies with animal concentration, weather, terrain, soils, and numerous other conditions. It is hoped that management practices found on dairy industry will benefit by the management practices. So properly applied the Management Practices, the factual study of dairy effluent by various physico-chemical characters concern for environmental health and safety.

Study Design: Effluent samples were collected from dairy industries. The samples were characterized by physical parameters like pH, temperature, TS, etc. and chemical parameters BOD, COD, DO etc.

Place and Duration of Study: Effluent samples were collected from dairy industry of district Kolhapur Maharashtra (India).Physico-chemical characteristics of the effluent during the months between March to August 2011.

Methodology: Total 4 samples of dairy effluents were collected by composite sampling at the time 9, 12, 3, 6 o’clock per day and stored at 4oc for further analysis. Then on the next day in quintet it was subjected to analyze the physicochemical parameters like Temperature, pH, DO, TDS, TSS, TS, BOD, COD, chloride, Sulphate, oil and Grease.

Results: The study revealed that the dairy effluent is slightly alkaline in nature, and high temperature, BOD & COD values obtained by the analysis of dairy effluents indicate the presence of heavy load of organic substances. Also a higher temperature and oils and Greases which lower the dissolved oxygen activities can cause serious problems in disposal of waste water. Above the standard value suspended and dissolved organic solids are responsible for creating nuisance.

Conclusion: Dairy industry tested in this study was found high levels  of pH, BOD, COD, TSS  It is very important that proper waste water treatment systems should be installed for the protection of the environmental health and for  the ecological balance.

Open Access Original Research Article

L-Phenylalanine and L-Tyrosine Catabolism by Thermophilic Geobacillus stearothermophilus

M. Afzal, S. Al-Awadhi, S. Oommen

Biotechnology Journal International, Page 581-591
DOI: 10.9734/BBJ/2013/4516

Aims: This study investigated the potential of soil thermophile Geobacillus stearothermophilus for the biotransformation of phenylalanine and tyrosine.

Study Design: G. Stearothermopilus grows well at 65ºC and has a good potential for transformation and biodegradation of many compounds including steroids, bile acids, tryptophan and other compounds. In this study G. stearothermophilus was harvested at mid-log phase at 65ºC, on tryptone yeast extract (TYE) medium. Cells were collected by centrifugation under aseptic conditions, washed with sterile water and suspended in phosphate buffer with phenylalanine or tyrosine as sole source of carbon at 65ºC.  Metabolic parameters were optimized for optimal growth of the organism utilizing aromatic amino acids as an exclusive source of carbon.

Methodology: The amino acid metabolites were exhaustively extracted with methanol from freeze dried broth. The concentrated pooled extracts were analyzed by thin layer chromatography (TLC) using polar solvent systems and purification of the extracts was achieved on preparative tlc plates and GC separations. The molecular structures of purified metabolites were established through spectral data.

Results: Sixteen metabolites of phenylalainine and seventeen metabolites of tyrosine were identified in this study. Tyrosine metabolism extensively produced melanin pigments that caused hitches in the purification of tyrosine metabolites. Tyr metabolites were analyzed in cells cultured for short time.

Conclusion: Our data suggest that G. stearothermophilus has a good potential to metabolize aromatic amino acids yielding hydroxylated, deaminated, decarboxylated and many other products. Oxidative metabolism of phenylalanine and tyrosine by a thermophilic G. stearothermophilus is being reported for the first time.

Open Access Original Research Article

Genetic Characterization of Insulin Growth Factor-1 and Its Receptor Genes in Egyptian Buffalo (Bubalus bubalis L.)

Othman E. Othman, Mohamed F. Abdel-Samad, Nadia A. Abo El Maaty, Karima M. Sewify

Biotechnology Journal International, Page 592-604
DOI: 10.9734/BBJ/2013/4869

Aim: The somatotropic axis (SA) comprises genes associated with economically important quantitative traits in livestock like mammary and muscle growth as well as carcass traits. Insulin growth factor-1 (IGF-1) and its receptor (IGF-1R) are two important genes belonging to the SA. The aim of this study was to evaluate the genetic polymorphism of IGF1/SnaBI and IGF-1R/TaqI restriction sites in Egyptian buffalo.

Methodology: Genomic DNA was extracted from blood samples of 100 healthy buffaloes maintained at the Mahlet Mussa and El-Gmeasa herds from 2010 to 2012. PCR was performed using primers flanking a 250-bp fragment of the regulatory region of the buffalo IGF-1 gene and a 616-bp fragment of the IGF-1R gene encompassing 51-bp from exon 12, 479-bp from intron 12 and 86-bp from exon 13. The PCR-amplified fragments were digested with SnaBI (IGF-1) and TaqI (IGF-1R), electrophoresed and analyzed on agarose gels stained with ethidium bromide. The two amplified fragments were also sequenced and aligned with published sequences.

Results: All buffaloes investigated in this study were genotyped BB (i.e., negative for the SnaBI restriction site at position 224^225 (TAC^GTA) of the IGF-1 regulatory region) and AA (i.e., positive for the TaqI restriction site at position 47^48 (T^CGA) of the IGF-1R exon 12).

Conclusion: Our Egyptian buffaloes were homozygous BB and AA for IGF1/SnaBI and IGF-1R/TaqI restriction sites, respectively. The nucleotide sequence data were submitted to NCBI/Bankit/GenBank with the accession numbers KC852883 and KC852884 for Egyptian buffalo IGF-1 and IGF-1R genes, respectively. The study of variation in these sequences may be useful in future marker-assisted selection (MAS) and genetic conservation programs.  

Open Access Review Article

Constraining the Pluripotent Fate of Human Embryonic Stem Cells for Tissue Engineering and Cell Therapy – The Turning Point of Cell-Based Regenerative Medicine

Xuejun H. Parsons

Biotechnology Journal International, Page 424-457
DOI: 10.9734/BBJ/2013/4309

To date, the lack of a clinically-suitable source of engraftable human stem/progenitor cells with adequate neurogenic potential has been the major setback in developing safe and effective cell-based therapies for regenerating the damaged or lost CNS structure and circuitry in a wide range of neurological disorders. Similarly, the lack of a clinically-suitable human cardiomyocyte source with adequate myocardium regenerative potential has been the major setback in regenerating the damaged human heart. Given the limited capacity of the CNS and heart for self-repair, there is a large unmet healthcare need to develop stem cell therapies to provide optimal regeneration and reconstruction treatment options to restore normal tissues and function. Derivation of human embryonic stem cells (hESCs) provides a powerful in vitro model system to investigate molecular controls in human embryogenesis as well as an unlimited source to generate the diversity of human somatic cell types for regenerative medicine. However, realizing the developmental and therapeutic potential of hESC derivatives has been hindered by the inefficiency and instability of generating clinically-relevant functional cells from pluripotent cells through conventional uncontrollable and incomplete multi-lineage differentiation. Recent advances and breakthroughs in hESC research have overcome some major obstacles in bringing hESC therapy derivatives towards clinical applications, including establishing defined culture systems for de novo derivation and maintenance of clinical-grade pluripotent hESCs and lineage-specific differentiation of pluripotent hESCs by small molecule induction. Retinoic acid was identified as sufficient to induce the specification of neuroectoderm direct from the pluripotent state of hESCs and trigger a cascade of neuronal lineage-specific progression to human neuronal progenitors and neurons of the developing CNS in high efficiency, purity, and neuronal lineage specificity by promoting nuclear translocation of the neuronal specific transcription factor Nurr-1. Similarly, nicotinamide was rendered sufficient to induce the specification of cardiomesoderm direct from the pluripotent state of hESCs by promoting the expression of the earliest cardiac-specific transcription factor Csx/Nkx2.5 and triggering progression to cardiac precursors and beating cardiomyocytes with high efficiency. This technology breakthrough enables direct conversion of pluripotent hESCs into a large supply of high purity neuronal cells or heart muscle cells with adequate capacity to regenerate CNS neurons and contractile heart muscles for developing safe and effective stem cell therapies. Transforming pluripotent hESCs into fate-restricted therapy derivatives dramatically increases the clinical efficacy of graft-dependent repair and safety of hESC-derived cellular products. Such milestone advances and medical innovations in hESC research allow generation of a large supply of clinical-grade hESC therapy derivatives targeting for major health problems, bringing cell-based regenerative medicine to a turning point