Fetal rhesus monkey lung cells can be grown in serum-free medium for the replication of dengue-2 vaccine virus

Summary Serum-free media were developed to grow diploid fetal rhesus monkey lung (DBS-FRhL-2) cells and to propagate dengue-type 2 virus vaccine strain PR-159 (dengue-2 vaccine virus). Vitamins, amino acids, growth factors, hormones and other organic compounds, and inorganic salts were substituted for fetal bovine serum. The composition of the medium that was optimal for growth of DBS-FRhL-2 cells differed from medium optimal for the propagation of dengue-2 vaccine virus. Insulin, epidermal growth factor, fibroblast growth factor, and platelet-derived growth factor were required for DBS-FRhL-2 cell proliferation in serum-free medium but were inhibitory for virus propagation. Adenosine, cytidine, guanosine, uridine, and thymidine, each at 0.01 mM concentration, were necessary as medium supplements to obtain a high yield of dengue-2 vaccine virus in DBS-FRhL-2 cells under serum-free conditions. DBS-FRhL-2 cells grown in serum-free medium produced dengue-2 vaccine virus with yields similar to those of cells grown in the presence of serum. Dengue-2 vaccine virus obtained under serum-free conditions retained its phenotypic markers such as temperature sensitivity and small plaque size. This investigation was supported by Contract DAMD-17-81-C1029 from the U.S. Army Medical Research and Development Command, and by The Hormel Foundation.     

Possibilities of vaccine manufacture in human diploid cell strains with a serum replacement factor.

Cell lines MDCK (canine kidney), BGM (Buffalo green monkey kidney) and human embryonic lung fibroblast will support viral growth efficiently in medium without serum. Both MRC-5 and WI-38 cell strains have been approved by the Food and Drug Administration for manufacturing viral vaccines against cytomegalovirus and varicella-zoster virus. In this study we examine these two cell lines and viruses for their ability to grow in medium containing a serum replacement. The serum substitute used is LPSR-1 (low protein serum replacement). Using LPSR-1 in defined medium, we demonstrate multipassage cell growth and viral cultivation and replication equivalent to those obtained in medium containing fetal bovine serum (FBS). Viral growth after complete elimination of FBS varies and depends on cell line and virus. Serum substitutes can eliminate FBS in the viral growth phase of vaccine production and reduce costs.     

Development and characerization of cell lines from subhuman primates

Summary Seven epithelial cell lines derived from kidney and 20 fibroblastic cell lines deriving from lung, heart, muscle, kidney, and skin tissue of five rhesus and six African green monkey fetuses have been established and propagated in culture. Four epithelial cell two fibroblastic cell lines resumed cell multiplication after a period of growth decline, and these lines developed cytogenetic changes and growth characteristics of cells capable of unlimited growth in vitro. Sixteen of the fibroblastic lines derived from lung, heart, muscle, or skin were characterized by a finite life consisting of a period of active cell multiplication, followed by growth decline, senescence, and cell death. Fibroblasts derived from lung appeared to have the greatest growth potential in terms of total population doublings, and fibroblastic lines from rhesus monkeys were usually capable of more doublings than similar lines from African green monkeys. All fibroblastic lines were predominantly diploid during active growth from passages 1 to 30, but several lines developed karyological changes preceding or during growth decline and senescence. All lines tested were found sensitive to a number of human viruses. All tests on these cells for microbial agents and for tumorigenicity have been negative, and the have been preserved by freezing without loss of properties. These cell lines may be useful as standardized substrates in studies requiring nonhuman primate cells. The research upon which this publication is based was performed pursuant to Contract No. NIH-69-100 with the Division of Biologics Standards of the National Institutes of Health.     

Effect of tunicamycin on cell fusion induced by Mason-Pfizer monkey virus.

Mason-Pfizer monkey virus, a D-type retrovirus, has been shown to induce multinucleate cell (syncytium) formation or cell fusion in several normal primate cells. A series of experiments has been carried out to examine whether a glycosylated "fusion-inducing" product is responsible for this biological property of Mason-Pfizer monkey virus. Treatment of rhesus monkey fetal lung cells with different concentrations of tunicamycin, a potent inhibitor of glycosylation, during infection with Mason-Pfizer monkey virus had no effect on cell fusion even though up to 5 micrograms of the drug per ml was tested. Furthermore, no significant effect on the extent of syncytium formation in rhesus monkey fetal lung cells was observed when the time of addition or duration of treatment with this inhibitor was varied. Nevertheless, tunicamycin was very effective in blocking glycosylation in rhesus cells since virions produced in the presence of this drug completely lacked gp70 and gp20, the two structural glycoproteins of Mason-Pfizer monkey virus. These non-glycosylated virus particles produced in the presence of tunicamycin were noninfectious as determined by a protein A binding assay and were unable to induce syncytium formation when assayed on rhesus cells. These results indicate that glycosylation of the fusion-inducing product is not required for multinucleate cell formation induced by Mason Pfizer monkey virus.     

Comparative sensitivity of various cell culture systems for isolation of viruses from wastewater and fecal samples.

In efforts to define the most sensitive cell culture systems for recovery of viruses from wastewaters, 181 samples were inoculated in parallel into tube cultures of various cell types and were plaqued in bottle and petri dish cultures of three types of monkey kidney cells. Polioviruses were recovered most frequently in the RD line of human rhabdomyosarcoma cells, group A coxsackieviruses in RD and human fetal diploid kidney (HFDK) cells, group B coxsackieviruses in the BGM line of African green monkey kidney cells, echoviruses in RD and primary rhesus monkey kidney (RhMK) cells, and reoviruses in RhMK cells. BGM cells were unsatisfactory for recovery of viruses other than polioviruses and group B coxsackieviruses, and a line of fetal rhesus monkey kidney (MFK) was not a satisfactory substitute for primary RhMK. With RhMK cells, comparable numbers of virus isolations were made in tube cultures and in plaque assays conducted in bottle cultures, but with BGM and MFK cells, fewer isolations were made by plaquing than by inoculation of tube cultures. In comparative plaque assays on fecal samples under three different overlays in bottle and plate cultures of RhMK, BGM, and MFK cells, it was found that plaquing in the most sensitive system, RhMK, was less efficient for virus recovery than was inoculation of tube cultures of RhMK or HFDK cells. Overall, plaque assays performed in petri dishes in a CO(2) incubator yielded fewer virus isolates than did parallel plaque assays performed in closed bottle cultures. Other limitations of plaque assays for recovery of human enteric viruses are discussed.     

Comparative Sensitivity of Various Cell Culture Systems for Isolation of Viruses from Wastewater and Fecal Samples

In efforts to define the most sensitive cell culture systems for recovery of viruses from wastewaters, 181 samples were inoculated in parallel into tube cultures of various cell types and were plaqued in bottle and petri dish cultures of three types of monkey kidney cells. Polioviruses were recovered most frequently in the RD line of human rhabdomyosarcoma cells, group A coxsackieviruses in RD and human fetal diploid kidney (HFDK) cells, group B coxsackieviruses in the BGM line of African green monkey kidney cells, echoviruses in RD and primary rhesus monkey kidney (RhMK) cells, and reoviruses in RhMK cells. BGM cells were unsatisfactory for recovery of viruses other than polioviruses and group B coxsackieviruses, and a line of fetal rhesus monkey kidney (MFK) was not a satisfactory substitute for primary RhMK. With RhMK cells, comparable numbers of virus isolations were made in tube cultures and in plaque assays conducted in bottle cultures, but with BGM and MFK cells, fewer isolations were made by plaquing than by inoculation of tube cultures. In comparative plaque assays on fecal samples under three different overlays in bottle and plate cultures of RhMK, BGM, and MFK cells, it was found that plaquing in the most sensitive system, RhMK, was less efficient for virus recovery than was inoculation of tube cultures of RhMK or HFDK cells. Overall, plaque assays performed in petri dishes in a CO₂ incubator yielded fewer virus isolates than did parallel plaque assays performed in closed bottle cultures. Other limitations of plaque assays for recovery of human enteric viruses are discussed.     

Mason-Pfizer Virus Characterization: a Similar Virus in a Human Amniotic Cell Line

Mason-Pfizer monkey virus (MP-MV) is a RNA virus with an RNA-instructed DNA polymerase first isolated from a rhesus monkey mammary adenocarcinoma in 1970. Until recently, there have been no other isolates. A continuous human amnion cell line, AO, was found to be producing a virus indistinguishable or closely related to the Mason-Pfizer virus as measured by morphological, immunological, and biochemical methods. By thin-section electron microscopy, the extracellular virus particle in AO line is 115 to 130 nm in diameter and has a preformed nucleoid (80 to 90 nm) before budding, properties which are also characteristic of MP-MV. Two proteins of the virus from the AO line were studied. By immunodiffusion, sera which react specifically with MP-MV give a line of identity with virus from the AO line. The AO viral RNA-instructed DNA polymerase purified by phosphocellulose chromatography was specifically inhibited by anti-MP-MV polymerase sera, and the AO cells contained both DNA and RNA sequences related to MP-MV (3)H-DNA. Viruses thus far indistinguishable from MP-MV have also recently been found by others in different human lines, raising again the question of the species of origin of MP-MV. Because the virus in the AO cells cannot be differentiated from MP-MV, we attempted to determine the origin of MP-MV virus by measuring DNA sequences related to MP-MV (3)H-DNA in uninfected human and rhesus monkey cells. The quantity of MP-MV-like DNA sequences in uninfected primate tissues was found to be much lower than the amount of DNA sequences of murine type-B or type-C viruses in uninfected murine tissues. Thus, it was not possible to determine whether the virus produced by AO cells or MP-MV was of human or monkey origin, or both.     

Susceptibility of cell lines of primate and nonprimate origin to certain human viruses

A comparative study was made of the susceptibility of 11 cell lines of human and animal origin, the WI-38 cell strain and fresh cultures of human thyroid, monkey kidney and hamster embryo tissues to certain human viruses. The animal cell lines were derived from monkey, rabbit, mouse, pig and calf tissues. The viruses used were strains of influenza A₂ and B viruses, parainfluenza viruses types 1, 2 and 3, RS virus, adenoviruses types 3, 4 and 21, poliovirus type 1 and Coxsackie A type 21 and Coxsackie B type 3 viruses. Cell lines derived from nonprimate tissues were generally less susceptible than cell cultures of human and simian origin. The combined use of fresh cultures of human thyroid and monkey kidney tissues and of a human cell line seems to provide a satisfactory indicator system for the viruses employed in this study.     

Replication of herpes simplex virus in two cell systems derived from rhesus monkeys

The replication of herpes simplex virus (HSV) in two cell systems derived from rhesus monkeys (LLC-MK2 and DBS-FRhL-2) was studied. In LLC-MK2, the growth of HSV-1 was abortive or extremely limited regardless of the multiplicity of infection, while that of HSV-2 was productive only on infection at high multiplicities. DBS-FRhL-2 cells supported growth of both types of HSV, although growth was highly dependent on the age of monolayers and the infectious dose of virus inocula. Plaques were produced in DBS-FRhL-2 cell monolayers inoculated with HSV-2 but not with HSV-1, although the efficiency of their formation in the former system was much less than in a system of FL and HSV-2. On the other hand, plaques were not produced in LLC-MK2 cell monolayers by either type of HSV. The growth of adapted variants of HSV-1 was also studied. In contrast to the parental strain, these variants replicated well in LLC-MK2 even at a low multiplicity of infection and produced clear plaques in the monolayers. Furthermore, persistent infections of HSV-2 were established in DBS-FRhL-2 cell monolayers under routine culture conditions.     

Infection of Human B Lymphocytes with Lymphocryptoviruses Related to Epstein-Barr Virus

Lymphocryptoviruses (LCVs) naturally infecting Old World nonhuman primates are closely related to the human LCV, Epstein-Barr virus (EBV), and share similar genome organization and sequences, biologic properties, epidemiology, and pathogenesis. LCVs can efficiently immortalize B lymphocytes from the autologous species, but the ability of a given LCV to immortalize B cells from other Old World primate species is variable. We found that LCV from rhesus monkeys did not immortalize human B cells, and EBV did not immortalize rhesus monkey B cells. In this study, baboon LCV could not immortalize human peripheral blood B cells but could readily immortalize rhesus monkey B cells. Thus, efficient LCV-induced B-cell immortalization across distant Old World primate species appears to be restricted by a species-specific block. To further characterize this species restriction, we first cloned the rhesus monkey LCV major membrane glycoprotein and discovered that the binding epitope for the EBV receptor, CD21, was highly conserved. Stable infections of human B cells with recombinant amplicons packaged in rhesus monkey or baboon LCV envelopes were also consistent with a species-restricted block occurring after virus binding and penetration. Transient infections of human B cells with simian LCV resulted in latent LCV EBNA-2 gene expression and activation of cell CD23 gene expression. EBV-immortalized human B cells could be coinfected with baboon LCV, and the simian virus persisted and replicated in human B cells. Thus, several lines of evidence indicate that the species restriction for efficient LCV-induced B-cell immortalization occurs beyond virus binding and penetration. This has important implications for the study of LCV infection in Old World primate models and for human xenotransplantation where simian LCVs may be inadvertently introduced into humans.     

Homogeneous cultivation of animal cells for the production of virus and virus products

Homogeneous technique facilitates the cultivation of large quantities of cells, reduces the risk of contamination by eliminating many manipulations, and makes practical the control of conditions such as pH and oxygen tension. Although most animal cells will not multiply in free suspension, certain cell lines have lost the requirement of being attached to a solid surface. These cells can be subcultured indefinitely but have some resemblance to cancer cells such as their abnormal karyotype. Certain cell linen developed from human embryonic tissue maintain their diploid character after repeated subculture and would seem to be ideal for the production of vaccines. However, strict regulations exist for viral products for human injection in that only cells taken from normal tissue and subcultured but once may be used. A microcarrier method in which cells adhere to DEAE-Sephadex beads permits a suspension culture which may be termed quasihomogeneous. The attached cells may be retained by sedimentation or by screening as the medium is replaced. Cell debirs from the original tissue is difficult to remove from microcarrier cultures; modifications of the trypsinization technique have alleviated but not solved this problem. Conditions for virus replication can be less critical than those for cell growth in that oxygen tension seems to have little influence on virus production. In cases where rate of virus production increases with specific growth rate of cells, homogeneous culture would have a advantage in maintaining a high cell mogeneous culture would have a valuble advantage in maintaining a high cell growth rate for a longer time. Some virus infections destroy cells, but others cause little change in cellular mteabolism except that virus is continually produced. The latter type can be conducted with a microcarrier in continuous culture with a virus titer exceeding 10⁷ plaque forming units per milliliter for over 50 days with Rubella-infected BHK cells.     

Identification of postentry restrictions to Mason-Pfizer monkey virus infection in New World monkey cells

TRIM5α has been shown to be a major postentry determinant of the host range for gammaretroviruses and lentiviruses and, more recently, spumaviruses. However, the restrictive potential of TRIM5α against other retroviruses has been largely unexplored. We sought to determine whether or not Mason-Pfizer monkey virus (M-PMV), a prototype betaretrovirus isolated from rhesus macaques, was sensitive to restriction by TRIM5α. Cell lines from both Old World and New World primate species were screened for their susceptibility to infection by vesicular stomatitis virus G protein pseudotyped M-PMV. All of the cell lines tested that were established from Old World primates were found to be susceptible to M-PMV infection. However, fibroblasts established from three New World monkey species specifically resisted infection by this virus. Exogenously expressing TRIM5α from either tamarin or squirrel monkeys in permissive cell lines resulted in a block to M-PMV infection. Restriction in the resistant cell line of spider monkey origin was determined to occur at a postentry stage. However, spider monkey TRIM5α expression in permissive cells failed to restrict M-PMV infection, and interference with endogenous TRIM5α in the spider monkey fibroblasts failed to relieve the block to infectivity. Our results demonstrate that TRIM5α specificity extends to betaretroviruses and suggest that New World monkeys have evolved additional mechanisms to restrict the infection of at least one primate betaretrovirus.     

Restriction of feline immunodeficiency virus by Ref1, Lv1, and primate TRIM5alpha proteins

The Ref1 and Lv1 postentry restrictions in human and monkey cells have been analyzed for lentiviruses in the primate and ungulate groups, but no data exist for the third (feline) group. We compared feline immunodeficiency virus (FIV) to other restricted (human immunodeficiency virus type 1 [HIV-1], equine infectious anemia virus [EIAV]) and unrestricted (NB-tropic murine leukemia virus [NB-MLV]) retroviruses across wide ranges of viral inputs in cells from multiple primate and nonprimate species. We also characterized restrictions conferred to permissive feline and canine cells engineered to express rhesus and human TRIM5alpha proteins and performed RNA interference (RNAi) against endogenous TRIM5alpha. We find that expression of rhesus or human TRIM5alpha proteins in feline cells restricts FIV, impairing pseudotyped vector transduction and viral replication, but rhesus TRIM5alpha is more restricting than human TRIM5alpha. Notably, however, canine cells did not support restriction by human TRIM5alpha and supported minimal restriction by rhesus TRIM5alpha, suggesting that these proteins may not function autonomously or that a canine factor interferes. Stable RNAi knockdown of endogenous rhesus TRIM5alpha resulted in marked increases in FIV and HIV-1 infectivities while having no effect on NB-MLV. A panel of nonprimate cell lines varied widely in susceptibility to lentiviral vector transduction, but normalized FIV and HIV-1 vectors varied concordantly. In contrast, in human and monkey cells, relative restriction of FIV compared to HIV-1 varied from none to substantial, with the greatest relative infectivity deficit for FIV vectors observed in human T-cell lines. Endogenous and introduced TRIM5alpha restrictions of FIV could be titrated by coinfections with FIV, HIV-1, or EIAV virus-like particles. Arsenic trioxide had complex and TRIM5alpha-independent enhancing effects on lentiviral but not NB-MLV infection. Implications for human gene therapy are discussed.     

Marsupial cells in long-term culture

Cell lines have been developed from several species of Australian marsupials and studied during long-term growth. Cell lines developed from macropodid skin or heart tissues all had reproducible finite life-spans. However, cell lines developed from dasyurids showed bariable behavior in culture: lines developed from Antechinus stuartii and Dasyurus viverrinus had finite life-spans, while lines developed from Sminthopsis crassicaudata had indefinite life-spans. S. crassicaudata lines usually became heteroplloid, but one was still diploid after 150 population doublings, while another contained a proportion (10%) of haploid cells. Other lines were developed from the peramelid, Perameles nasuta, and the phanlngerid, Trichosurus vulpecula.     

The level of CD4 expression limits infection of primary rhesus monkey macrophages by a T-tropic simian immunodeficiency virus and macrophagetropic human immunodeficiency viruses

The entry of primate immunodeficiency viruses into cells is dependent on the interaction of the viral envelope glycoproteins with receptors, CD4, and specific members of the chemokine receptor family. Although in many cases the tropism of these viruses is explained by the qualitative pattern of coreceptor expression, several instances have been observed where the expression of a coreceptor on the cell surface is not sufficient to allow infection by a virus that successfully utilizes the coreceptor in a different context. For example, both the T-tropic simian immunodeficiency virus (SIV) SIVmac239 and the macrophagetropic (M-tropic) SIVmac316 can utilize CD4 and CCR5 as coreceptors, and both viruses can infect primary T lymphocytes, yet only SIVmac316 can efficiently infect CCR5-expressing primary macrophages from rhesus monkeys. Likewise, M-tropic strains of human immunodeficiency virus type 1 (HIV-1) do not infect primary rhesus monkey macrophages efficiently. Here we show that the basis of this restriction is the low level of CD4 on the surface of these cells. Overexpression of human or rhesus monkey CD4 in primary rhesus monkey macrophages allowed infection by both T-tropic and M-tropic SIV and by primary M-tropic HIV-1. By contrast, CCR5 overexpression did not specifically compensate for the inefficient infection of primary monkey macrophages by T-tropic SIV or M-tropic HIV-1. Apparently, the limited ability of these viruses to utilize a low density of CD4 for target cell entry accounts for the restriction of these viruses in primary rhesus monkey macrophages.     

Immunofluorescence Staining of Group B Coxsackieviruses

Studies were conducted on the sensitivity and specificity of indirect fluorescent-antibody (FA) staining for identification of group B coxsackieviruses. Antisera produced in four different species (monkeys, rabbits, horses, hamsters) and immune ascitic fluids prepared in mice were compared for suitability in FA staining. The horse antisera showed high titers of nonspecific staining, and the rabbit antisera showed relatively low homologous FA titers. Immune reagents from monkeys, hamsters, and mice were used for homologous and heterologous testing against cell cultures infected with the various group B coxsackieviruses. Antisera or immune ascitic fluids produced in these three species showed some heterotypic and nonspecific staining at low dilutions, with the monkey antisera showing the highest heterotypic titers. However, the immune reagents could be diluted to a point where they gave no heterotypic reactivity, but still showed characteristic homotypic staining. Heterotypic staining appeared as diffuse, low-level staining of the cells, whereas homotypic staining revealed characteristic, brightly staining aggregates of viral antigen in the cytoplasm of the infected cells. By using hamster immune sera, appropriately diluted to eliminate heterotypic staining and yet give strong homotypic staining, it was possible to identify correctly 79 (93%) of 85 field strains of group B coxsackieviruses at the first passage level in BS-C-1 cells; the remainder of the strains were identified after two passages in BS-CS-1 cells. No incorrect identifications were made. A limited number of field strains of group B coxsackieviruses were passed into rhesus monkey kidney and human fetal diploid kidney cells, and these were all correctly identified by FA staining, even the strains which failed to produce a cytopathic effect in the human fetal diploid kidney cells. Two human heart and brain tissues from which coxsackievirus type B4 had been isolated failed to show homotypic FA staining in excess of nonspecific or heterotypic staining.     

Virus Susceptibility of a New Simian Cell Line of Fetal Origin

The cultivation and characterization of a cell line derived from the foreskin of a fetal, rhesus monkey (rhfs2) are described. This cell line has proven satisfactory for isolation and assay of a variety of viral agents of human and simian origin. Virus titrations performed on foreskin cells yielded titers comparable to, or higher than, those obtained in rhesus monkey kidney cells (LLC-MK2). Replicate isolation attempts in our laboratory from simian clinical specimens have proven rhfs2 superior to LLC-MK2 for ease of detection and frequency of isolation.     

Establishment, characterization, and viral susceptibility of two cell lines derived from goldfish Carassius auratus muscle and swim bladder

Goldfish Carassius auratus are common aquarium fish and have a significant economic and research value, having considerable worth to fisheries as a baitfish and the ability to adapt to a range of habitats. Two cell lines were established from goldfish muscle and swim bladder tissue, in order to create a biological monitoring tool for viral diseases. Cell lines were optimally maintained at 30 degrees C in Leibovitz-15 medium supplemented with 20% fetal bovine serum. Propagation of goldfish cells was serum dependent, with a low plating efficiency (>16%). Karyotyping analysis indicated that both cell lines remained diploid, with a mean chromosomal count of 104. Results of viral challenge assays revealed that both cell lines shared similar patterns of viral susceptibility and production to infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, snakehead rhabdovirus, and spring viremia carp virus. Both cell lines demonstrated a higher sensitivity and significantly larger viral production than control brown bullhead cells for channel catfish virus. These newly established cell lines will be used as a diagnostic tool for viral diseases in this fish species and also for the isolation and study of goldfish viruses in the future.     

Comparative effects of human platelet growth factor on the growth and morphology of human fetal and adult diploid fibroblasts

Human platelet growth factor (HPGF) caused marked growth and morphological responses in two lines of fetal and two lines of adult human diploid skin fibroblasts. Dose-response studies demonstrated differences between these two cell types. Adult cells required ten times more HPGF than fetal cells for optimal growth. In the presence of HPGF both cell lines decreased three- to four-fold in size, and their morphology in stationary culture changed to an extremely long, thin fibroblastic shape; reduction in size was accompanied by a corresponding decrease in total protein and fatty acid-containing lipid. These decreases were directly related to the concentration of HPGF in the growth medium. The results suggest that human cells in different stages of cellular development have different requirements for growth-promoting agents (HPGF) and that the use of HPGF on human diploid fibroblasts provides an excellent model system to study growth-associated changes in intermediary metabolism.     

Challenges of primate embryonic stem cell research

Embryonic stem (ES) cells hold great promise for treating degenerative diseases, including diabetes, Parkinson's, Alzheimer's, neural degeneration, and cardiomyopathies. This research is controversial to some because producing ES cells requires destroying embryos, which generally means human embryos. However, some of the surplus human embryos available from in vitro fertilization (IVF) clinics may have a high rate of genetic errors and therefore would be unsuitable for ES cell research. Although gross chromosome errors can readily be detected in ES cells, other anomalies such as mitochondrial DNA defects may have gone unrecognized. An insurmountable problem is that there are no human ES cells derived from in vivo-produced embryos to provide normal comparative data. In contrast, some monkey ES cell lines have been produced using in vivo-generated, normal embryos obtained from fertile animals; these can represent a "gold standard" for primate ES cells. In this review, we argue a need for strong research programs using rhesus monkey ES cells, conducted in parallel with studies on human ES and adult stem cells, to derive the maximum information about the biology of normal stem cells and to produce technical protocols for their directed differentiation into safe and functional replacement cells, tissues, and organs. In contrast, ES cell research using only human cell lines is likely to be incomplete, which could hinder research progress, and delay or diminish the effective application of ES cell technology to the treatment of human diseases.