Requirements for human cardiomyocytes

‘Requirements for human cardiomyocytes'', jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research, is the first guideline for human cardiomyocytes in China. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packing requirements, storage requirements, transportation requirements and waste disposal requirements for human cardiomyocytes, which is designed to normalize and standardize human cardiomyocyte research and production. It was originally released by the China Society for Cell Biology on 9 January 2021. We hope that the publication of this guideline will promote institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human cardiomyocytes for applications.     

Necessity for two human chromosomes for human chorionic gonadotropin production in human-mouse hybrids

Through a series of human-mouse hybrids we have identified that two human chromosomes, 10 and 18, must be present for production of the pregnancy protein hormone human chorionic gonadotropin (hCG). Human choriocarcinoma cells producing hCG were hybridized to mouse cells. From 49 independent clones three hybrid clones continued to produce whole hCG. Chromosomal analysis was done on the 3 producer clones and 5 nonproducer clones. The additional 41 nonproducer clones were genetically characterized by isozymes. Only when chromosomes 10 and 18 were present in a clone would the whole hCG molecule be produced. Clones with only 10 or only 18 did not produce hormone. Nine subclones of a producer clone confirmed this observation. Three subclones retaining both 10 and 18 continued to produce hCG. This study demonstrated the need to use cellular chromosome data and population enzyme data to identify two chromosomes necessary for hCG production in heterogeneous human-mouse hybrids.     

A human hybrid myeloma for production of human monoclonal antibodies

We produced somatic cell hybrids between human myeloma cells and a lymphoblastoid cell line that is hypoxanthine phosphoribosyl transferase-deficient and ouabain-resistant. These hybrids were phenotypically similar to the human myeloma parental cells and grew as well as the human lymphoblastoid parental cells. After counterselection in 6-thioguanine, mutants that were 6-thioguanine-and ouabain-resistant were obtained, one of which was used as a fusion partner with lymphoblastoid B cells that produce anti-tetanus toxoid (TT) antibodies. These hybrids secreted human anti-TT monoclonal antibodies in much larger amounts than the parental lymphoblastoid cells, and were stable for a period of over 10 mo until the present time. Thus, by hybridizing plasmacytomas with lymphoblastoid cells, we constructed a fusion partner that secretes large amounts of immunoglobulin (Ig), grows at a fast rate, has a high fusion frequency, and supports the production of monoclonal antibodies over long periods of time. Moreover, anti-TT antibody-producing hybrids have been grown as solid tumors in irradiated BALB/c nude mice and then adopted to ascites growth, producing 1 to 8 mg of human immunoglobulin per 1 ml of ascites fluid.     

Purification techniques for human interferons

This article outlines the development and general status of present purification methods for human interferons. The isolation of each interferon species from natural sources is extremely difficult because of molecular characteristics, high losses of activity and the small amount of interferon protein present in production media. Few of the highly sophisticated methods meet the requirements for scale-up or give acceptable recoveries for a production process. The adaptation of purification procedures to the different interferon species is described, such as initial concentration, the extraction of beta interferon (IFN-β) by aqueous two-phase systems and the final purification of alpha interferon (IFN-α) and beta interferon to homogeneity. H.p.l.c. techniques are discussed in more detail together with problems in the purification of beta interferon and gamma interferon (IFN-γ). The range of interferon expression and excretion in recombinant microbial and animal cells is reviewed and different approaches for the solubilization and purification of intracellular recombinant interferons are described, which are covered mainly in patent applications.     

Radioimmunoassay for human salivary amylase

A radioimmunoassay (RIA) for human salivary amylase was developed. Human salivary amylase was purified from parotid saliva by a combination of Sephadex gel filtration and cation exchange chromatography. Purified salivary amylase was used both as the standard antigen and for the generation of ¹²⁵I-labeled amylase. Antibody to salivary amylase was raised in New Zealand white rabbits and used in a nonequilibrium double-antibody procedure for the RIA. The RIA was sensitive (10 ng/ml) and specific, displaying a limited cross-reactivity for pancreatic amylase (1%, $\text{w}\text{w}$). Analysis of patient sera by RIA shows that salivary amylase constitutes approximately 60% of the total serum amylase, that the salivary amylase found in the serum of patients with Sjögren's syndrome and macroamylasemia is immunologically indistinguishable from that of normal persons, and that salivary amylase can be evaluated by RIA in the serum of patients with pancreatitis.     

Requirments for primary human hepatocyte

‘Requirements for Primary Human Hepatocyte’ is the first set of guidelines on Primary Human Hepatocyte in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements and transportation requirements for Primary Human Hepatocyte, which is applicable to the quality control for Primary Human Hepatocyte. It was originally released by the China Society for Cell Biology on 9 January 2021. We hope that publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols and accelerate the international standardization of Primary Human Hepatocyte for applications.     

Insect cells for human food

There is a need for novel protein sources. Insects are a possible interesting source of protein. They are nutritious in terms of protein (40-75 g/100g dry weight) and minerals. Insect protein is of high quality and has a high digestibility (77-98%) and concentration of essential amino acids (46-96% of the nutritional profile). Also insect cells may be a promising novel source of protein. Choice of cell line, growth conditions and use of the baculovirus expression system opens up possibilities to engineer the nutritional value of the biomass. The technological limits as well as consumer acceptance of insect cell based food remains to be investigated.     

Cell receptors for human adenoviruses

During the early stage of the adenovirus infection, the virion binds to a "primary receptor" on the host cell plasma membrane via the fibre projection jetting out of the penton base capsomers located at the twelve apices of the icosahedral capsid. The second step consists of a receptor-mediated endocytosis which involves membrane integrin molecules (the "secondary receptors") and the RGD and/or LDV motifs of penton base. The latter step is inhibited at low temperature, whereas virus attachment to its primary receptor is temperature-independent. Two different primary receptors with a high affinity for the Adenovirus have been recently identified. One is common to Coxsackievirus B3 and adenovirus (CAR), the other one corresponds to a conserved region of the alpha-2 domain of the heavy chain of the major histocompatibility complex class I molecules (MHC-I-alpha 2), overlapping tryptophane-167. The receptor usage by the virus is governed by both cellular and viral parameters. On the cellular side, the relative abundance of one versus the other type of primary receptors would theoretically determine the virus choice: CAR receptor has been mainly found in tissues from mesodermic origin, whereas MHC-I-alpha 2 is ubiquitous. On the virus side, the molecular determinants of the receptor usage have been mapped to the terminal knob of the fiber projection, and have been found to be different for CAR and MHC-I-alpha 2. CAR recognizes linear motifs in fiber knobs in a subgroup-dependent manner, as it binds to all Adenovirus serotypes except for the subgroup B members. MHC-I-alpha 2 however recognizes conformational epitopes carried by fiber knobs from all serotypes tested including subgroup B members. These results should have significant implications in the cell targeting of adenoviral vectors used in gene therapy.     

Collection protocol for human pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation. Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing β-cells followed by lower proportions of glucagon-containing α-cells and somatostatin-containing δ-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes. Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.     

Biomarkers for human radiation exposure

There is a concern over the potential use of radioactive isotopes as a weapon of terror. The detonation of a radiation dispersal device, the so-called “dirty bomb” can lead to public panic. In order to estimate risks associated with radiation exposure, it is important to understand the biological effects of radiation exposure. Based on this knowledge, biomarkers to monitor potentially exposed populations after a radiological accident can be developed and would be extremely valuable for emergency response. While the traditional radiation exposure biomarkers based on cytogenetic assays serve as standard, the development of rapid and noninvasive tests for radiation exposure is needed. The genomics based knowledge is providing new avenues for investigation. The examination of gene expression after ionizing radiation exposure could serve as a potential molecular marker for biodosimetry. Microarray based studies are identifying new radiation responsive genes that could potentially be used as biomarkers of human exposure to radiation after an accident.     

Measurement standards for human metacarpals

Standards for measuring the metacarpals are absent from commonly used osteometric guides. Perhaps the closest to a set of standard measurements in common use today are those proposed by Scheuer and Elkington (Scheuer and Elkington: J Forensic Sci 38 (1993) 769–788) for forensic sex assessment. They include caliper measurements of interarticular length, base and head width, base and head height, and maximum midshaft diameter. Over the last decade, a new set of measurements that encompass similar dimensions to those used by Scheuer and Elkington, but which are taken with a mini‐osteometric board (MOB) have been developed by the lead author. Use of the MOB avoids the need to manipulate both the bone and calipers in three‐dimensional space and causes less strain on the hands. However, the question of intra‐ and interobserver accuracy has not been adequately addressed for either set of measurements. The purpose of this study was to test both the Scheuer/Elkington and MOB measurements on 20 hands from 10 anatomical skeletons for intra‐ and inter‐observer accuracy. The study found that 92% of the MOB measures had a lower intraobserver error, and 88% had a lower interobserver error than did the caliper measurements. It also found that the maximum midshaft diameter measurement used by Scheuer and Elkington is more repeatable than a mediolateral diameter. Overall, 88% of the 25 MOB measurements had median intraobserver error rates of under 1.5%, compared with 60% of the caliper measurements. Furthermore, the MOB measurements as a set were taken 10 to 12% faster than the caliper measurements. Am J Phys Anthropol 157:322–329, 2015. © 2015 Wiley Periodicals, Inc.     

Artificial gravity for human missions

The author examines the merits and problems associated with full-time rotation and intermittent G stimulation as weightlessness countermeasures during prolonged space flight.