Skin care efficacy study of recombinant humanized collagen based on in vitro level

Studying the skin care efficacy of recombinant humanized collagen based on in vitro level.The stability of the recombinant humanized collagen was first analyzed by treating at different temperatures,then its skincare efficacy based on in vitro level was evaluated by detecting the inhibition rate of elastase,the inhibition rate of collagenase,the protein content of type I collagen in human fibroblasts,the inhibition of reactive oxygen species(ROS)with human keratinocytes,and the effects of the recombinant humanized collagen on the expression of hyaluronic acid(HA),filaggrin(FLG)and transglutaminase 1(TGM1)in keratinocytes.The results showed that recombinant humanized collagen was able to maintain stability at temperatures below 70℃.With regard to its skincare efficacy,recombinant humanized collagen could inhibit elastase and collagenase activities and promote the increase of type I collagen content in human fibroblasts.It also showed good inhibition of ROS in keratinocytes in vitro and could increase the expression of HA,FLG,and TGM1 in keratinocytes.In short,the recombinant humanized collagen exhibited a favourable skin care effect in vitro level.This study proved that it has potential firming,anti-wrinkle,moisturizing,and repairing efficacy,and is a valuable cosmetic raw material.

Commentary on:“Human neural stem cell-derived artificial organelles to improve oxidative phosphorylation”

Mitochondrial function is fundamental to neuroregeneration,particularly in neurons,where high energy demands are essential for repair and recovery(Patrón and Zinsmaier,2016;Beckervordersandforth et al.,2017;Iwata et al.,2023).Mitochondrial dysfunction,characterized by an imbalance in ATP levels and excessive production of mitochondrial reactive oxygen species,is a key factor that impedes neural regeneration in neurodegenerative diseases and after neuronal injury(Han et al.,2016,2020;Zheng et al.,2016;Zong et al.,2024).

Human endogenous retrovirus type-W and multiple sclerosis–related smoldering neuroinflammation

Introduction to human endogenous retrovirus type-W(HERV-W): Genomic inheritance from the past includes retroviral sequences that have been stably incorporated into our genomes and account for up to 8% of human DNA.

The Combined Effect of Lumenato and Ceramide in the Protection of Collagen Damage Induced by Neutrophils in Normal Human Dermal Fibroblasts

Introduction: Collagen is the primary structural protein fibroblasts produce in the skin’s extracellular matrix. Infiltration of neutrophils into the epidermis and dermis by exposure to UV causes collagen damage and contributes to photoaging. Methods: To study the combined effect of Lumenato and ceramide in preventing collagen-1 damage induced by phagocytes, we used co-cultures of normal human dermal fibroblasts (fibroblasts) and activated human neutrophils. The present study aimed to determine the protective effect of the combination of Lumenato and ceramide on fibroblast collagen-1 damage induced by neutrophils. Results: Lumenato (in the range of 6.5 - 208 μg/ml) or ceramide (in the range of 0.1 - 50 μM) inhibited the production of superoxides and MPO by TNFα-stimulated neutrophils, as well as the production of NO by LPS-stimulated macrophages in a dose-dependent manner. The combinations of Lumenato and ceramide, in low concentrations, caused synergistic prevention of fibroblasts’ collagen-1 damage induced by TNFα-activated neutrophils, detected by fluorescence immunostaining and WB analysis. MPO activity in the supernatants of the co-cultures was also synergistically inhibited. Adding Lumenato or ceramide singly or in combinations in these low concentrations to the fibroblast cultures did not affect the expression of collagen-1. The combinations of Lumenato or ceramide in these concentrations also caused a synergistic inhibition of NO production by activated macrophages. Conclusions: The results suggest that combining low concentrations of Lumenato and ceramide results in synergistic protection against fibroblasts’ collagen-1 damage induced by neutrophils, thus indicating their possible potential for enhanced skin health.

Treatment of spinal cord injury with biomaterials and stem cell therapy in non-human primates and humans

Spinal cord injury results in the loss of sensory,motor,and autonomic functions,which almost always produces permanent physical disability.Thus,in the search for more effective treatments than those already applied for years,which are not entirely efficient,researches have been able to demonstrate the potential of biological strategies using biomaterials to tissue manufacturing through bioengineering and stem cell therapy as a neuroregenerative approach,seeking to promote neuronal recovery after spinal cord injury.Each of these strategies has been developed and meticulously evaluated in several animal models with the aim of analyzing the potential of interventions for neuronal repair and,consequently,boosting functional recovery.Although the majority of experimental research has been conducted in rodents,there is increasing recognition of the importance,and need,of evaluating the safety and efficacy of these interventions in non-human primates before moving to clinical trials involving therapies potentially promising in humans.This article is a literature review from databases(PubMed,Science Direct,Elsevier,Scielo,Redalyc,Cochrane,and NCBI)from 10 years ago to date,using keywords(spinal cord injury,cell therapy,non-human primates,humans,and bioengineering in spinal cord injury).From 110 retrieved articles,after two selection rounds based on inclusion and exclusion criteria,21 articles were analyzed.Thus,this review arises from the need to recognize the experimental therapeutic advances applied in non-human primates and even humans,aimed at deepening these strategies and identifying the advantages and influence of the results on extrapolation for clinical applicability in humans.

Effects of P301L-TAU on post-translational modifications of microtubules in human iPSC-derived cortical neurons and TAU transgenic mice

TAU is a microtubule-associated protein that promotes microtubule assembly and stability in the axon.TAU is missorted and aggregated in an array of diseases known as tauopathies.Microtubules are essential for neuronal function and regulated via a complex set of post-translational modifications,changes of which affect microtubule stability and dynamics,microtubule interaction with other proteins and cellular structures,and mediate recruitment of microtubule-severing enzymes.As impairment of microtubule dynamics causes neuronal dysfunction,we hypothesize cognitive impairment in human disease to be impacted by impairment of microtubule dynamics.We therefore aimed to study the effects of a disease-causing mutation of TAU(P301L)on the levels and localization of microtubule post-translational modifications indicative of microtubule stability and dynamics,to assess whether P301L-TAU causes stability-changing modifications to microtubules.To investigate TAU localization,phosphorylation,and effects on tubulin post-translational modifications,we expressed wild-type or P301L-TAU in human MAPT-KO induced pluripotent stem cell-derived neurons(i Neurons)and studied TAU in neurons in the hippocampus of mice transgenic for human P301L-TAU(p R5 mice).Human neurons expressing the longest TAU isoform(2N4R)with the P301L mutation showed increased TAU phosphorylation at the AT8,but not the p-Ser-262 epitope,and increased polyglutamylation and acetylation of microtubules compared with endogenous TAU-expressing neurons.P301L-TAU showed pronounced somatodendritic presence,but also successful axonal enrichment and a similar axodendritic distribution comparable to exogenously expressed 2N4R-wildtype-TAU.P301L-TAU-expressing hippocampal neurons in transgenic mice showed prominent missorting and tauopathy-typical AT8-phosphorylation of TAU and increased polyglutamylation,but reduced acetylation,of microtubules compared with non-transgenic littermates.In sum,P301L-TAU results in changes in microtubule PTMs,suggestive of impairment of microtubule stability.This is accompanied by missorting and aggregation of TAU in mice but not in i Neurons.Microtubule PTMs/impairment may be of key importance in tauopathies.

Spatial transcriptomics combined with single-nucleus RNA sequencing reveals glial cell heterogeneity in the human spinal cord

Glial cells play crucial roles in regulating physiological and pathological functions,including sensation,the response to infection and acute injury,and chronic neurodegenerative disorders.Glial cells include astrocytes,microglia,and oligodendrocytes in the central nervous system,and satellite glial cells and Schwann cells in the peripheral nervous system.Despite the greater understanding of glial cell types and functional heterogeneity achieved through single-cell and single-nucleus RNA sequencing in animal models,few studies have investigated the transcriptomic profiles of glial cells in the human spinal cord.Here,we used high-throughput single-nucleus RNA sequencing and spatial transcriptomics to map the cellular and molecular heterogeneity of astrocytes,microglia,and oligodendrocytes in the human spinal cord.To explore the conservation and divergence across species,we compared these findings with those from mice.In the human spinal cord,astrocytes,microglia,and oligodendrocytes were each divided into six distinct transcriptomic subclusters.In the mouse spinal cord,astrocytes,microglia,and oligodendrocytes were divided into five,four,and five distinct transcriptomic subclusters,respectively.The comparative results revealed substantial heterogeneity in all glial cell types between humans and mice.Additionally,we detected sex differences in gene expression in human spinal cord glial cells.Specifically,in all astrocyte subtypes,the levels of NEAT1 and CHI3L1 were higher in males than in females,whereas the levels of CST3 were lower in males than in females.In all microglial subtypes,all differentially expressed genes were located on the sex chromosomes.In addition to sex-specific gene differences,the levels of MT-ND4,MT2A,MT-ATP6,MT-CO3,MT-ND2,MT-ND3,and MT-CO_(2) in all spinal cord oligodendrocyte subtypes were higher in females than in males.Collectively,the present dataset extensively characterizes glial cell heterogeneity and offers a valuable resource for exploring the cellular basis of spinal cordrelated illnesses,including chronic pain,amyotrophic lateral sclerosis,and multiple sclerosis.

Activation of adult endogenous neurogenesis by a hyaluronic acid collagen gel containing basic fibroblast growth factor promotes remodeling and functional recovery of the injured cerebral cortex

The presence of endogenous neural stem/progenitor cells in the adult mammalian brain suggests that the central nervous system can be repaired and regenerated after injury.However,whether it is possible to stimulate neurogenesis and reconstruct cortical layers II to VI in non-neurogenic regions,such as the cortex,remains unknown.In this study,we implanted a hyaluronic acid collagen gel loaded with basic fibroblast growth factor into the motor cortex immediately following traumatic injury.Our findings reveal that this gel effectively stimulated the proliferation and migration of endogenous neural stem/progenitor cells,as well as their differentiation into mature and functionally integrated neurons.Importantly,these new neurons reconstructed the architecture of cortical layers II to VI,integrated into the existing neural circuitry,and ultimately led to improved brain function.These findings offer novel insight into potential clinical treatments for traumatic cerebral cortex injuries.

Collagen scaffold combined with human umbilical cord-mesenchymal stem cells transplantation for acute complete spinal cord injury

Currently, there is no effective strategy to promote functional recovery after a spinal cord injury. Collagen scaffolds can not only provide support and guidance for axonal regeneration, but can also serve as a bridge for nerve regeneration at the injury site. They can additionally be used as carriers to retain mesenchymal stem cells at the injury site to enhance their effectiveness. Hence, we hypothesized that transplanting human umbilical cord-mesenchymal stem cells on collagen scaffolds would enhance healing following acute complete spinal cord injury. Here, we test this hypothesis through animal studies and a phase I clinical trial.(1) Animal experiments: Models of completely transected spinal cord injury were established in rats and canines by microsurgery. Mesenchymal stem cells derived from neonatal umbilical cord tissue were adsorbed onto collagen scaffolds and surgically implanted at the injury site in rats and canines;the animals were observed after 1 week–6 months. The transplantation resulted in increased motor scores, enhanced amplitude and shortened latency of the motor evoked potential, and reduced injury area as measured by magnetic resonance imaging.(2) Phase I clinical trial: Forty patients with acute complete cervical injuries were enrolled at the Characteristic Medical Center of Chinese People's Armed Police Force and divided into two groups. The treatment group(n = 20) received collagen scaffolds loaded with mesenchymal stem cells derived from neonatal umbilical cordtissues;the control group(n = 20) did not receive the stem-cell loaded collagen implant. All patients were followed for 12 months. In the treatment group, the American Spinal Injury Association scores and activities of daily life scores were increased, bowel and urinary functions were recovered, and residual urine volume was reduced compared with the pre-treatment baseline. Furthermore, magnetic resonance imaging showed that new nerve fiber connections were formed, and diffusion tensor imaging showed that electrophysiological activity was recovered after the treatment. No serious complication was observed during follow-up. In contrast, the neurological functions of the patients in the control group were not improved over the follow-up period. The above data preliminarily demonstrate that the transplantation of human umbilical cord-mesenchymal stem cells on a collagen scaffold can promote the recovery of neurological function after acute spinal cord injury. In the future, these results need to be confirmed in a multicenter, randomized controlled clinical trial with a larger sample size. The clinical trial was approved by the Ethics Committee of the Characteristic Medical Center of Chinese People's Armed Police Force on February 3, 2016(approval No. PJHEC-2016-A8). All animal experiments were approved by the Ethics Committee of the Characteristic Medical Center of Chinese People's Armed Police Force on May 20, 2015(approval No. PJHEC-2015-D5).

Chicken collagen hydrolysates differentially mediate anti-inflammatory activity and type I collagen synthesis on human dermal fibroblasts

Collagen is a major extracellular matrix protein.Given the potential anti-inflammatory and antioxidant profiles of these bioactive compounds,there has been increasing interest in using collagen derived peptides and peptide-rich collagen hydrolysates for skin health,due to their immunomodulatory,antioxidant and proliferative effects on dermal fibroblasts.However,all hydrolysates are not equally effective in exerting the beneficial effects;hence,further research is needed to determine the factors that improve the therapeutic applicability of such preparations.We used different enzymatic conditions to generate a number of different collagen hydrolysates with distinct peptide profiles.We found that the use of two rather than one enzyme for hydrolysis generates a greater abundance of low molecular weight peptides with consequent improvement in bioactive properties.Testing these hydrolysates on human dermal fibroblasts showed distinct actions on inflammatory changes,oxidative stress,type I collagen synthesis and cellular proliferation.Our findings suggest that different enzymatic conditions affect the peptide profile of hydrolysates and differentially regulate their biological activities and potential protective responses on dermal fibroblasts.

Stability improvement of human collagenα1(I)chain using insulin as a fusion partner

To enhance the stability of recombinant human collagen α1(I) chains(rhCOL1 A1) in production and purification stages, a gene fragment fusing COL1 A1 and insulin protein coding domains was synthesized and inserted into the pPIC9 K expression vector. The fusion peptide-expressing Pichia pastoris strain was created by transformation.After optimization of shake flask cultures, the ultimate intracellular expression level of the insulin-collagen α1(I) chain fusion protein(INS-COL1 A1) reached about 300 mg·L^(-1), and no obvious protein degradation was found in the fermentation and purification processes. The His-tagged recombinant fusion protein was detected by western blotting and was effectively purified using Ni^(2+)-chelating chromatography. A prominent improvement in the stability of INS-COL1 A1 was observed compared to rhCOL1 A1 in vitro, and the rhCOL1 A1 released from the fusion protein was studied by LC–MS/MS and in bioassays. The results showed that the purified rhCOL1 A1 was consistent with the native protein in amino acid composition and had a similar biological compatibility. To our knowledge, this is the first study to demonstrate the use of insulin as a fusion protein to improve the stability of easily degradable proteins.

Herbal compound 861 regulates mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells

AIM： To identify the role of herbal compound 861 （Cpd 861） in the regulation of mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells （HSCs）. METHODS： mRNA levels of collagen types I and III, matrix metalloproteinase 1 （MMP-1）, matrix metalloproteinase 2 （MMP-2）, membrane type-1 matrix metalloproteinase （MT1-MMP）, tissue inhibitor of metalloproteinase 1 （TIMP-1）, and transforming growth factor β1 （TGF-β1） in cultured-activated HSCs treated with Cpd 861 or interferon-γ, （IFN-γ,） were determined by real-time PCR. RESULTS： Both Cpd 861 and IFN-γ reduced the mRNA levels of collagen type Ⅲ, MMP-2 and TGF-β1. Moreover, Cpd 861 significantly enhanced the MMP-1 mRNA levels while down-regulated the TIMP-1 mRNA expression, increasing the ratio of MMP-1 to TIMP-1 to （6.3 ＋ 0.3）- fold compared to the control group. CONCLUSION： The anti-fibrosis function of Cpd 861 may be mediated by both decreased interstitial collagen sythesis by inhibiting the transcription of collagen type Ⅲ and TGF-β1 and increased degradation of these collagens by up-regulating MMP-1 and down-regulating TIMP-1 mRNA levels.

Effects of fibril- or fixed-collagen on matrix metalloproteinase-1 and tissue inhibitor of matrix metalloproteinase-1 production in the human hepatocyte cell line HLE

AIM:Matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) are central to the spontaneous resolution of liver fibrosis. The mechanisms involved have been investigated in hepatic stellate cells (HSC), but not in hepatocytes. We investigated the effects of fibril- and fixed-collagen on MMP-1 and TIMP-1 production in hepatocytes, using the HLE cell line. METHODS: Fibril type I and IV collagen were prepared by HCI digestion of type I and IV collagen, respectively. For fixed-collagen, culture dishes were coated with fibril type I or IV collagen and fixed by ultraviolet. Type I collagenase activity was measured using fluorescein isothiocyanate-labeled type I collagen. MMP-1 and TIMP-1 in HLE cells were measured by a one-step sandwich enzyme immunoassay. RESULTS: Both fibril type I and IV collagen significantly increased type I collagenase activity about two-fold compared with no fibril collagen. The effects of the fibril collagen were not affected by the coating condition. There was no significant difference in the effects on collagenase activity between cells cultured in medium containing fibril type I collagen and those cultured in the presence of type IV collagen. Both types of fibril collagen significantly increased MMP-1 production, and showed more than 10-fold higher levels of MMP-1 than the control. The enhanced MMP-1 production by fibril collagens was unaffected by the coating condition. By contrast, TIMP-1 production was not changed by the addition of fibril type I or IV collagen, and neither was it affected by the coating conditions. Coating with type I collagen significantly suppressed MMP-1 production by almost one-tenth compared with no coating. By contrast, TIMP-1 production was not affected by either the absence of a collagen coat or by increasing the concentration of the coating collagen. CONCLUSION: These results indicated that, in HLE cells, fibril- and fixed-collagen have opposite effects on MMP-1 production without affecting TIMP production. Fibril collagen induced collagenase activity by up-regulation of MMP-1 production without affecting TIMP-1 production. By contrast, fixed collagen reduced MMP-1 production. Our results suggest that hepatocytes might also play an important role in the regulation of the hepatic fibrosis alongside HSC.

INFLUENCE OF QUERCETIN AND X-RAY ON COLLAGEN SYNTHESIS OF CULTURED HUMAN KELOID-DERIVED FIBROBLASTS

Objoctive To investigate the effects of quercetin and X-ray on collagen synthesis of cultured human keloid-derived fibroblast and the mechanism. Methods Collagen synthesis of cultured human keloid and normal fibroblasts were detected by hydroxyproline colorimetric analysis. Immunocytochemical staining was used to investigate collagen I and III expression, mRNA expression of collagen Ⅰ and Ⅲ, and transforming growth factor （ TGF）-β1 were assayed by reverse transcription-polymerase chain reaction （RT-PCR） and real-time PCR. Results Quercetin inhibited the collagen synthesis of both keloid and normal fibroblasts in a dose-dependent man- ner. Immunocytochemical staining indicated that collagenⅠ and Ⅲ were down-regulated by quercetin and X-ray （P 〈 0.05 ）, particularly collagen I （ P 〈 0. 05 ）. mRNA expression of both collagen I and III in quercetin groups significantly decreased compared with that in control group （ P 〈 0. 05 ）, especially in the group treated with both quercetin and X-ray （ P 〈 0. 01 ）. mRNA level of TGF-[31 gene was down-regulated by quercertin （ P 〈 0. 05 ）. Conclusions Quercetin will probably be one of the new medicines which could effectively treat keloid. Quercetin combined with X-ray could reduce the dose of radiation.

A Pilot Study of Trophic Level and Human Origins at the Xiaoshuangqiao Site, China(ca. 1400 BC) Using 8D Values of Collagen

We present here a pilot study to examine trophic level effects and migration patterns at the middle Shang Dynasty site of Xiaoshuangqiao in Henan Province using δD results combined with δ^13C and δ^15N values. A total of 33 specimens（17 humans, 7 cattle, 5 pigs, 3 sheep, 1 dog） of bone collagen were isotopically analyzed for δ^13C, δ^15N, and dD values. A strong positive correlation（R^2 = 0.94）between mean δ^15N and δD values of herbivores（cattle and sheep）, omnivores（pig）, carnivores（dog）and humans was observed. The δD results were found to increase by -10‰ to 20‰ from herbivores to omnivores to carnivorous, evidence that collagen δD results are a useful indicator for the study of trophic levels and dietary patterns at archaeological sites. The δD results were also used to examine the origins of two different groups of individuals buried at Xiaoshuangqiao. Individuals buried in sacrificial pits of district V had mean δD values（-47.0 ± 2.9‰, n = 11） that were significantly（p =0.049） elevated compared to the people buried in the stratigraphy of district IX（-51.3 ± 3.3‰, n = 3）,indicating that they were ingesting water from different locations. In addition, the D values of the people buried in the stratigraphy were similar to the pigs（-54.5 ± 4.2‰, n = 5） at Xiaoshuangqiao,suggesting that they were most probably of the local population, and that the individuals buried in the sacrificial pits were most possibly from the coast and prisoners of the Dongyi（“东夷”）people. Thus,δD results have the potential to examine human origins and migration patterns and should be increasingly used in conjunction with δ^13C and δ^15N values at archaeological sites.

Optimization of Fermentation Process for Human-like Collagen Production of Recombinant Escherichia coli Using Response Surface Methodology

In order to improve the production of human-like collagen III(HLC III)by fed-batch culture of recombinant Escherichia coli BL21,the Plackett-Burman and Box-Behnken design were applied to optimize the fermentation process parameters.Three variables(induction time,inoculum age and pH),which have significant effects on HLC III production,were selected from eight variables by Plackett-Burman design.With the regression coefficient analysis in the Box-Behnken design,a relationship between HLC III production and three significant factors was obtained,and the optimum levels of the three variables were as follows:induction time 3.2h,inoculum age 12.6 h and pH 6.7.The 3D response surface plots and 2D contour plots created by the Box-Behnken design showed that the interaction between induction time and pH and that between innoculum age and pH were significant.An average 9.68 g·L1HLC III production was attained in the validation experiment under optimized condition,which was 80%higher than the yield of 5.36 g·L1before optimization.

Process Control for Production of Human-like Collagen in Fed-batch Culture of Escherichia coli BL 21

Recombinant E. coli BL 21 was cultivated in high cell density to produce human-like collagen. The effects of the feeding of nitrogen source, controlled by an auto on/off-feeding mode with two different cycles of 0.5min and 4min intervals, oxygen-enrichment methods and inducement strength on the cell yield and human-like collagen production were investigated. The studies showed that nitrogen source feeding in fast cycle could result in higher human-like collagen production than that in slow cycle; and the feedback regulation of glucose, increase of the pressure of fermentation bioreactor, and supply of oxygen-enriched air could all increase cell yield and human-like collagen production. The effects of inducement strength on protein expression were found important. When OD600 reached 90-100, the cultivation temperature was increased to 42℃ to begin induction for 2-3 h, and then shifted to 39℃ for 5-6h induction, the cell density and human-like collagen production could reach 96g·L-1 [DCW (dry cell mass)] and 19.8% (g·g-1 DCW) respectively.

Analysis of Metabolic Products by Response Surface Methodology for Production of Human-like Collagen II

Recombinant Escherichia coli BL21 is used to produce human-like collagen. The key constituents of media are optimized using response surface methodology (RSM). Before thermal induction, the highest biomass production and the lowest production of some hazardous by-products, especially acetic acid, were obtained in the media containing 0.085 mol·L-1 glucose and 0.019 mol·L-1 nitrogen (carbon-nitrogen ratio, 4.47:1). After thermal induction, when the concentrations of glucose and nitrogen in the media were 0.065 mol·L-1 and 0.017 mol·L-1 , respectively (carbon-nitrogen ratio, 3.82:1), the productivity of human-like collagen per cell was the highest while that of acetic acid was the lowest. The extended analysis showed that the production of lactic acid and propionic acid increased while that of some intermediate acids of the tricarboxylic acid cycle decreased if the dose of glucose increased.

Enrichment of putative human epidermal stem cells based on cell size and collagen type IV adhesiveness

The enrichment and identification of human epidermal stem cells （EpSCs） are of paramount importance for both basic research and clinical application. Although several approaches for the enrichment of EpSCs have been established, enriching a pure population of viable EpSCs is still a challenging task. An improved approach is worth developing to enhance the purity and viability of EpSCs. Here we report that cell size combined with collagen type IV adhesiveness can be used in an improved approach to enrich pure and viable human EpSCs. We separated the rap- idly adherent keratinocytes into three populations that range in size from 5-7 μm （population A）, to 7-9 μm （population B）, to ≥9μm （population C） in diameter, and found that human putative EpSCs could be further enriched in population A with the smallest size. Among the three populations, population A displayed the highest density of plintegrin receptor, contained the highest percentage of cells in G0/G1 phase, showed the highest nucleus to cytoplasm ratio, and possessed the highest colony formation efficiency （CFE）. When injected into murine blastocysts, these cells participated in multi-tissue formation. More significantly, compared with a previous approach that sorted putative EpSCs according to pl-integrin antibody staining, the viability of the EpSCs enriched by the improved approach was significantly enhanced. Our results provide a putative strategy for the enrichment of human EpSCs, and encourage further study into the role of cell size in stem cell biology.

Kinetics of High Cell Density Fed-batch Culture of Recombinant Escherichia coli Producing Human-like Collagen

The kinetics of batch and fed-batch cultures of recombinant Escherichia coli producing human-like collagen was investigated. In the batch culture, a kinetic model of a simple growth-association system was concluded without consideration of cell endogeneous metabolism. The cell lag time, the maximum specific growth rate and Yx/s were determined as 1.75h, 0.65h^-1 and 0.51g·g^-1, respectively. In the fed-batch culture, different specific growth rates were set at （0.15, 0.2, 0.25h^-1） by the method of pseudo-exponential feeding, and the expressions for the specific rate of substrate consumption, the growth kinetics and the product formation kinetics of each phase were obtained. The result shows that the concentrations of cell and product can reach 77.5g·L^-1 and 10.2g·L^-1 respectively. The modal predictions are in good agreement with the experimental data.