Journal of Science and Applications: Biomedicine

6-19

Authors: Shaida Moghaddassi, Will Eyestone, Colin E. Bishop

The annual demand for therapeutic human serum albumin (HSA) is estimated to be more than 500 metric tons worldwide. As a major protein in the human body, HSA plays a vital role in many physiological processes, including the maintenance of oncotic pressure and the transportation of various biomolecules and pharmaceuticals. Currently, all HSA used for clinical blood expansion purposes is isolated from pooled human blood or plasma, an unpredictably fluctuating supply that can at times fall to dangerously low levels. Furthermore, this supply is derived from thousands of different donors and can potentially result in the spread of pathogenic contaminants to recipients. The use of transgenic animals, such as cattle, as living bioreactors, provides a potential solution to this problem by enabling the large-scale production of recombinant HSA (rHSA) in a cost-effective manner. Cattle are capable of producing large amounts of milk that can potentially yield abundant quantities of a desired recombinant protein. The production of rHSA in the milk of cattle would provide an economical resource that circumvents the current dependence on blood-derived sources. One challenge to this system, however, is the presence of endogenous bovine serum albumin (BSA) in the milk. BSA is a highly conserved ortholog of HSA that necessitates a tedious and prohibitively expensive purification process, which has up to now hindered the efficient purification of rHSA from bovine milk. Here we overview our approach to humanize the endogenous BSA gene, replacing it with an rHSA minigene construct, which should allow for normal expression of rHSA protein in the liver, as well as exogenous expression of rHSA in the milk. The future generation of such cattle supports the potential for a safer and more reliable source of therapeutic rHSA.

1-5

Authors: Zhenlong Wang, Li Xue, Ziming Wang, Weimin Gan, Haiwen Chen, Hecheng Li, Peng Zhang, Hongliang Li, Tie Chong

Our objective is to evaluate the effect and safety of transurethral resection of prostate (TURP) and U100 laser lithotripsy for benign prostate hyperplasia patients with complicating bladder lithiasis. 79 cases with bladder outflow obstruction (BOO) secondary to benign prostate hyperplasia (BPH) complicating bladder lithiasis were collected. Based on the different ways of bladder lithiasis lithotripsy, the patients were randomized into two groups: the punch lithotrite group (43 cases) and the U100 laser group (42 cases). All patients were assessed perioperatively and followed at one month postoperatively. The preoperative and postoperative parameters included international prostate symptom score (IPSS), maximum urinary flow rates (Qmax), postvoid residual urine (PVRU) volume, patient baseline characteristics, perioperative data, and postoperative outcomes. In the two groups, the IPSS scores and the maximum flow rate (MFR) showed significant differences when compared to the preoperative data (P﹤0.05). The stone-free rate (97.2%) in the U100 laser group was significantly higher than that in the punch group (62.8%), with a statistical difference (P＜0.05). For stones ﹤1.5 cm, the time of lithotripsy and stone-free rate in the U100 laser group was not statistically different as compared with those in the punch group. For stones >2.5 cm, the lithotripsy efficiency and the stone-free rate in the U100 laser group increased significantly with a statistical difference when compared with those in the punch group. In conclusion, the therapeutic effects of the two lithotripsy approaches showed no difference for calculi with a diameter <1.5cm. While for calculi with a diameter >2.5cm, the U100 laser lithotripsy was recommended. We have shown that the U100 laser lithotripsy, when combined with TURP, is an effective and safe treatment for BPH with bladder lithiasis.

24-33

Authors: Fengjuan Yao, Guohua Yin, Ruiguo Cai, Yingqin Luo, Zhiqiang Yu, and Jichao Yuan

Coixlacryma-jobi L. is a very useful medicine and widely used for various human diseases such as abdominal tumors, cancer, and respiratory tract infections. However, its growth and yield response to different photoperiods has not been studied yet. In this study, three periods (three-leaf, five-leaf, and eight-leaf) and two shading treatment periods (8 and 11 hours daylight) were designed to investigate the effects of photoperiod on the growth and yield of Coix cultivars in Sichuan China. Four cultivars were selected and obtained from Beijing, Shandong, Nanjing, and Ya’an, respectively. Several agronomic characteristics of Coix, such as tillers, effective tillers, leaf number, plant height, and yield traits, were recorded. The results indicated that Beijing cultivar was more sensitive to light than other cultivars on the short-day growth rate. Southern cultivars show earlier daylight sensitivity than northern cultivars, while daylight length of the northern cultivars was longer than that of southern cultivars. For plant height, photoperiod had more impact on Coix at the earlier period than the late period. The three-leaf period was more sensitive to daylight than the five- and eight-leaf period, and the longer shading treatment of the three-leaf period promoted Coix to produce more tillers. Our study reveals that shading treatment shortens the growth of Coix and can accelerate the transition from nutritive to reproductive growth. It was found that photoperiod had a significant influence on the growth of Coix but exhibited no influence on its yield.

1-7

Authors: Guoguang Niu, Tracy Criswell, Etai Sapoznik, Sang-Jin Lee, Shay Soker

Electrospun scaffolds fabricated from poly(epsilon-caprolactone) (PCL)/collagen composite are promising candidates for vascular graft applications. In order to improve the biocompatibility and mechanical strength of PCL/collagen scaffolds, two different cross-linking methods were applied and compared: (1) 2.5% glutaraldehyde (GA) vapor cross-linking for 6 h and (2) genipin (GN) cross-linking for 3 days in 70% ethanol. Scaffolds cross-linked with GN demonstrated higher cross-linking degree, collagen content, tensile strength and a similar stiffness compared with GA cross-linked scaffolds. Scaffolds cross-linked with GA and GN both supported endothelial and smooth muscle cell attachment and growth. However, cells seeded on GN cross-linked scaffolds displayed a faster growth rate and deeper penetration into the scaffolds than those cross-linked with GA. These data suggest that the use of GN to cross-link PCL/collagen scaffolds may enhance the cellularization of vascular scaffolds resulting in a more rapid development of integrated functional vessels in vivo.

15-23

Authors: J. Koudy Williams, Silmi Mariya, Anthony Atala, Shay Soker

Regenerative medicine promises to address the organ shortage by engineering replacement cells, tissues and organs using adult, progenitor, or human embryonic stem cells. However, embryonic stem cells can form teratomas and bone marrow-derived progenitor cells have limited expansion capabilities. We recently described a subpopulation of c-kit+ human amniotic fluid-derived stem (hAFS) cells that are highly expansive, do not form teratomas and can yield differentiated cell derivatives corresponding to the three germ layers. To this end, and because nonhuman primates (NHP) represent a critical step towards pre-clinical testing of the AFS technology, the goal of this study was to determine if AFS cells could be isolated from amniotic samples of monkeys. Expansive cell cultures were derived from 12/28 amniotic fluid samples of cynomolgus monkey. Cells were isolated and expanded in Chang’s media and c-kit+ cells were isolated, further expanded and clonal populations obtained. The putative AFS cells were characterized as to their karyotype, ability to expand, identification of cell-surface and nuclear markers (qPCR) and ability to differentiate to mesodermal lineages such as bone, muscle, fat, cartilage and endothelial cells. Highly expansive multipotent cells were isolated most readily from the second trimester of pregnancy (8/15 samples, or 53%), and less so from either the first (1/3, or 33%), or third (3/10, or 30%) trimester. Cell were diploid in all cases and were consistently expanded through at least 20 passages (10 passages for the 3rd trimester cells) without losing their proliferation potential, as confirmed by cell cycle analysis (G1, G2, S phase) and doubling times. The AFS cells had low-level expression of pluripotentiality markers (Oct4, Nanog, SOX2) compared to NHP embryonic stem cells, while expressing markers similar to that of adult mesodermal-lineage cells (CD44, CD90, CD73 and CD105). nhpAFS cells express the major, but not minor, histocompatability antigen and were consistently differentiated into adipogenic, osteogenic, myogenic, chondrocytic and endothelial lineages. Because of their robust expansion capabilities and ability to differentiate into several mesodermal lineages, it is concluded that nhpAFS cells, similar to hAFS cels, have the characteristics needed to bioengineer a wide-variety of mesodermal-cell containing organs and tissues.