Long-term Primary Cultures Research Articles

Development of long-term primary cell culture of Macrobrachium rosenbergii: morphology, metabolic activity, and cell-cycle analysis

This study describes our attempts to generate a sustainable cell culture of Macrobrachium rosenbergii. We present here a continuous longitudinal study on the embryonic primary cell culture of freshwater prawn M. rosenbergii that was uniquely monitored for up to 90 days with regard to its morphology, metabolic activity, and cell-cycle parameters. The daily monitoring of cells’ wellbeing and morphology showed seeded cells to be changing from attached singular diverse-sized cells after days 4–10 to interconnected clusters of cells, which apparently increased in number as detected by their density in the well. Moreover, the cultures demonstrated an autonomous transition during days 7–10, from completely two-dimensional (2D) morphology to a combination of 2D and three-dimensional (3D) growing structures, leading to the formation of multilayered spheroid-like cell masses. The metabolic activity of cultures showed a non-linear elevated pattern peaking on day 26, demonstrating proliferation and increment in the number of cells, retaining statistically significant elevated metabolic activity up to 40 days, and thereafter gradually declining. In parallel, cell-cycle analyses performed through florescence-activated cell sorting (FACS) showed that the G0/G1 and S phases were inversely proportional to each other. Proliferation, based on metabolic activity, in the cultures was sustained by a significant increase in the portion of cells arrested in the S phase, from day 4 up to day 24, and then a decrease between days 45 and 90. Sorting the populations in the M. rosenbergii primary embryonic cell culture on days 3 and 24 revealed eight seeded populations, most of them expressing the putative proliferation markers MrMYC and MrPCNA, while six of them expressed also the putative stem-cell markers MrOct-4 and MrSox-2/3. Therefore, assuming the increment in cell density and metabolic activity and the reduction in G0/G1 distribution toward S, as well as the increment in G2/M, all pointing toward proliferation, we further hypothesized that splitting the cultures along the experiment at the high-proliferating mitotic ratio peaks would enable successful passages. Indeed, in prawn embryonic primary cell culture, we succeeded in executing two consecutive passages: the first after 8 days in culture and the second 4 days following the first passage. Cells after both passages expressed the species-specific Mr18S, along with the proliferative markers MrMYC and MrPCNA and the stem-cell markers MrOct-4 and MrSox-2/3. After several decades of research efforts to establish a crustacean cell line—with no published success—here, we present M. rosenbergii cultures composed of putative proliferating/stem-cell subpopulations or appearing like clones. These mix-population prawn embryonic primary cell cultures could serve as a basic platform for immortalization and contribute to the long-term goal of establishing sustainable cell-culture lines.

Next-Generation Modeling of Cancer Using Organoids.

In the last decade, organoid technology has become a cornerstone in cancer research. Organoids are long-term primary cell cultures, usually of epithelial origin, grown in a three-dimensional (3D) protein matrix and a fully defined medium. Organoids can be derived from many organs and cancer types and sites, encompassing both murine and human tissues. Importantly, they can be established from various stages during tumor evolution and recapitulate with high accuracy patient genomics and phenotypes in vitro, offering a platform for personalized medicine. Additionally, organoids are remarkably amendable for experimental manipulation. Taken together, these features make organoids a powerful tool with applications in basic cancer research and personalized medicine. Here, we will discuss the origins of organoid culture, applications in cancer research, and how cancer organoids can synergize with other models of cancer to drive basic discoveries as well as to translate these toward clinical solutions.

A novel medium for long-term primary culture of hemocytes of Metapenaeus ensis

The development of a suitable shrimp cell medium is essential for achieving a long-term cell culture and finite cell line of shrimps routinely. In this study, we have successfully developed an optimal shrimp cell medium that can be used for long-term in vitro culture and continuous subculture of the hemolymph cells (or hemocytes) of greasyback shrimp Metapenaeus ensis, designated as MeH cells, by shrimp serum-based and supplements-based optimization of the basic and growth medium. In this article, we have focused on the details for the preparation of the optimal shrimp cell medium by diluting and mixing of various stock solutions as well as the methods for isolation and primary culture of MeH cells.•A novel shrimp cell growth medium is developed for long-term shrimp hemocytes culture.•The preparation method of shrimp cell growth medium is successfully established.•Obvious cell activity and proliferation potential of isolated shrimp cells can be maintained beyond 30 days.

Routine development of long-term primary cell culture and finite cell line from the hemolymph of greasyback shrimp (Metapenaeus ensis) and virus susceptibility

Long-term and continuous shrimp cell cultures can offer an indispensable tool for studies on the isolation, multiplication and pathogenic mechanism of shrimp causative viruses at cellular and molecular levels. However, they are hard to be developed routinely and continuous shrimp cell line is still unavailable. This can be attributed to a great degree to the unsuitability of medium used. In this study, we have successfully developed a long-term culture condition for the hemolymph cells (or hemocytes) of greasyback shrimp Metapenaeus ensis, designated as MeH cells, by shrimp serum-based and supplements-based optimization of the basic and growth medium. Using the newly developed gelatin-containing optimal basic medium (SBM-12), MeH cell monolayer could be routinely developed and maintained healthily beyond 275 days. In the novel optimal growth medium (SGM-10), the proliferation potentials of MeH cells have been markedly improved, evidenced by the presence of dividing cells in the primarily cultured 30-day hemocytes, suggesting that the mitosis-arrest of MeH cells has been tentatively broken. It was also found that among the 4 types of hemocytes (granulocyte, semi-granulocyte, hyalinocyte and prohaemocyte), the hyalinocytes could not survive after a long-term in vitro culture, but the prohaemocytes could survive well and thus could serve as a good cell source. The addition of gelatin could significantly alter the attachment and cellular morphology of MeH cells and result in a loose attachment and round shape, which made it easy for the cells to be passaged by physical flushing for up to 32 times and a finite shrimp cell line can be developed routinely. Virus susceptibility test showed that the MeH cells cultured in the optimal growth medium were sensitive to shrimp covert mortality nodavirus (CMNV), and obvious cytopathic effects and viral multiplication were detected in the infected cells. In a word, the success in the routine development of long-term primary cell culture and finite cell line from shrimps in this study will greatly facilitate the establishment of shrimp continuous cell lines and studies on shrimp viruses.

Transcriptomic Profile of Genes Regulating the Structural Organization of Porcine Atrial Cardiomyocytes during Primary In Vitro Culture.

Numerous cardiovascular diseases (CVD) eventually lead to severe myocardial dysfunction, which is the most common cause of death worldwide. A better understanding of underlying molecular mechanisms of cardiovascular pathologies seems to be crucial to develop effective therapeutic options. Therefore, a worthwhile endeavor is a detailed molecular characterization of cells extracted from the myocardium. A transcriptomic profile of atrial cardiomyocytes during long-term primary cell culture revealed the expression patterns depending on the duration of the culture and the heart segment of origin (right atrial appendage and right atrium). Differentially expressed genes (DEGs) were classified as involved in ontological groups such as: “cellular component assembly”, “cellular component organization”, “cellular component biogenesis”, and “cytoskeleton organization”. Transcriptomic profiling allowed us to indicate the increased expression of COL5A2, COL8A1, and COL12A1, encoding different collagen subunits, pivotal in cardiac extracellular matrix (ECM) structure. Conversely, genes important for cellular architecture, such as ABLIM1, TMOD1, XIRP1, and PHACTR1, were downregulated during in vitro culture. The culture conditions may create a favorable environment for reconstruction of the ECM structures, whereas they may be suboptimal for expression of some pivotal transcripts responsible for the formation of intracellular structures.

Erratum to: Fatty acids related genes expression undergo substantial changes in porcine oviductal epithelial cells during long-term primary culture

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Propagation of CJD Prions in Primary Murine Glia Cells Expressing Human PrPc.

There are various existing cell models for the propagation of animal prions. However, in vitro propagation of human prions has been a long-standing challenge. This study presents the establishment of a long-term primary murine glia culture expressing the human prion protein homozygous for methionine at codon 129, which allows in vitro propagation of Creutzfeldt–Jakob disease (CJD) prions (variant CJD (vCJD) and sporadic CJD (sCJD) type MM2). Prion propagation could be detected by Western blotting of pathological proteinase K-resistant prion protein (PrPSc) from 120 days post exposure. The accumulation of PrPSc could be intensified by adding a cationic lipid mixture to the infectious brain homogenate at the time of infection. Stable propagation of human prions in a long-term murine glia cell culture represents a new tool for future drug development and for mechanistic studies in the field of human prion biology. In addition, our cell model can reduce the need for bioassays with human prions and thereby contributes to further implementation of the 3R principles aiming at replacement, reduction and refinement of animal experiments.

Establishment of Long-Term Primary Cortical Neuronal Cultures From Neonatal Opossum Monodelphis domestica.

Primary dissociated neuronal cultures have become a standard model for studying central nervous system (CNS) development. Such cultures are predominantly prepared from the hippocampus or cortex of rodents (mice and rats), while other mammals are less used. Here, we describe the establishment and extensive characterization of the primary dissociated neuronal cultures derived from the cortex of the gray South American short-tailed opossums, Monodelphis domestica. Opossums are unique in their ability to fully regenerate their CNS after an injury during their early postnatal development. Thus, we used cortex of postnatal day (P) 3–5 opossum to establish long-surviving and nearly pure neuronal cultures, as well as mixed cultures composed of radial glia cells (RGCs) in which their neurogenic and gliogenic potential was confirmed. Both types of cultures can survive for more than 1 month in vitro. We also prepared neuronal cultures from the P16–18 opossum cortex, which were composed of astrocytes and microglia, in addition to neurons. The long-surviving opossum primary dissociated neuronal cultures represent a novel mammalian in vitro platform particularly useful to study CNS development and regeneration.

New Gene Markers Involved in Molecular Processes of Tissue Repair, Response to Wounding and Regeneration Are Differently Expressed in Fibroblasts from Porcine Oral Mucosa during Long-Term Primary Culture.

Simple SummaryWound healing and vascularization mechanisms are key steps in the complex morphological process of tissue reconstruction. Additionally, these processes in the oral cavity are more rapid than in the skin and result in less scar formation. Epithelial cells and fibroblasts play an important role in the process of wound healing. In our study, we focused on fibroblasts and monitored changes in gene expression during their in vitro culture. Based on the analysis, we distinguished three groups of processes that play important roles in tissue regeneration: response to wounding, wound healing and vascularization. We identified genes that were involved in all three processes. These genes could be selected as tissue specific repair markers for oral fibroblasts.The mechanisms of wound healing and vascularization are crucial steps of the complex morphological process of tissue reconstruction. In addition to epithelial cells, fibroblasts play an important role in this process. They are characterized by dynamic proliferation and they form the stroma for epithelial cells. In this study, we have used primary cultures of oral fibroblasts, obtained from porcine buccal mucosa. Cells were maintained long-term in in vitro conditions, in order to investigate the expression profile of the molecular markers involved in wound healing and vascularization. Based on the Affymetrix assays, we have observed three ontological groups of markers as wound healing group, response to wounding group and vascularization group, represented by different genes characterized by their expression profile during long-term primary in vitro culture (IVC) of porcine oral fibroblasts. Following the analysis of gene expression in three previously identified groups of genes, we have identified that transforming growth factor beta 1 (TGFB1), ITGB3, PDPN, and ETS1 are involved in all three processes, suggesting that these genes could be recognized as markers of repair specific for oral fibroblasts within the porcine mucosal tissue.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons.

Parkinson's disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

The genes regulating maintenance of cellular protein location are differentially expressed in porcine epithelial oviductal cells during longterm in vitro cultivation

Abstract The oviduct is a part of female reproductive tract that is essential for successful fertilization and early embryo development. It is lined with epithelium consisting of two types of cells: ciliated and secretory. The primary function of ciliated oviductal epithelial cells (OECs) is to support the transport of gametes and embryos through the ovary, whereas secretory OECs produce components of the oviductal fluid. Undoubtedly, the oviductal epithelium plays a major part in the early aspects of pregnancy development, from providing an optimal environment for gametes and embryos to supporting fertilization. Therefore, our aim was to gain a better insight into the genetic changes underlying function of these cells. We have harvested OECs from crossbred gilts (n=45), at the age of about nine months and which displayed two regular estrous cycles, and established long-term primary culture of porcine OECs. Microarray analysis was utilized to determine differentially expressed genes during day 1, 7, 15 and 30 of cultivation, with our results revealing54 differentially expressed genes belonging to three ontology groups: „maintenance of location”, „maintenance of protein location” and „maintenance of protein location in cell”. Since the biochemistry and morphology of epithelial cells may change during long term cultivation, we conclude that our results are a reflection of these changes and help to shed a light on porcine OECs properties in in vitro environment. Running title: Maintenance of cellular protein location in porcine epithelial oviductal cells

Genes regulating biochemical pathways of oxygen metabolism in porcine oviductal epithelial cells during long-term IVC

Abstract Oxygen metabolism has an important role in the normal functioning of reproductive system, as well as the pathogenesis of female infertility. Oxidative stress seems to be responsible for the initiation or development of reproductive organ diseases, including polycystic ovary syndrome, endometriosis, preeclampsia, etc. Given the important role of maintaining balance between the production of ROS and antioxidant defence in the proper functioning of reproductive system, in the present study we aimed to analyse the expression of genes related to oxygen metabolism in porcine oviductal epithelial cells during long-term in vitro culture. The oviducts were collected from 45 crossbred gilts at the age of approximately nine months that displayed at least two regular oestrous cycles. The oviductal endothelial cells were isolated by enzymatic digestion to establish long-term primary cultures. Gene expression changes between 7, 15 and 30 daysof culturewere analysed with the use ofwhole transcriptome profiling by Affymetrix microarrays. The most of the “cellular response to oxidative stress” genes were upregulated. However, we did not observe any main trend in changes within the “cellular response to oxygen-containing compound” ontology group, where the gene expression levels were changed in various manner. Running title: Oxygen metabolism in porcine oviductal epithelial cells

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo.

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Long-term culture and characterization of patient-derived primary hepatocytes using conditional reprogramming.

Cultivation of primary human hepatocytes (PHHs) often faces obstacles including failure of long-term in vitro culture, weak proliferation ability, rapid loss of liver-specific function and morphology, and tendency of fibrosis. Previous research focused on immortalization methods, such as telomerase and viral, to culture immortalized primary human hepatocytes, which may lose some of the normal properties. However, non-immortalized PHHs often fail to maintain long-term viability and functionality. These highlight the urgent need for developing new culture strategy for PHHs. In the present study, we isolated PHHs from fresh human liver tissues representing different liver diseases and age groups. We used conditional reprogramming, without permanent immortalization, for long-term in vitro primary human hepatocytes cultivation and characterization. For functional characterization, we assessed CYP3A4, 1A1 and 2C9 activities and measured the mRNA expression of albumin, s100a4, krt8, krt18, cyp1a1, cyp3a4, cyp2b6, cyp2c8, cyp2c9, and cyp2d6. Additionally, we compared the DNA fingerprint of the cells against their original liver tissues using short tandem repeat (STR) analysis. We found that PHHs-derived from young patients can survive for more than three months, while the lifespan of primary human hepatocytes derived from adult patients ranges from two to three months, which is longer than most commercial primary hepatocytes. Importantly, the cells at early passages retain strong CYP3A4, 1A1 and 2C9 activities and the DNA fingerprints are identical with their original tissues. Through conditional programming, we achieved, for the first time, a high level of success rate in the long-term in vitro cultivation of primary human hepatocytes-derived patients representing diverse liver disease. Moreover, the conditional programming cell culture technology reported in this paper requires neither co-culture with additive cells, nor complex and expensive components, such as collagen sandwich or spheroid culture. We thus believe that the patient-derived PHHs cultivation using conditional programming may provide a viable and valuable cell model to study liver disease-related mechanisms. Impact statement Commercially available primary human hepatocytes rapidly lose their proliferative ability and liver-specific functions over a few cultivation days. The demand for pharmaceutical toxicity screening and liver disease research requires the development of long-term primary hepatocyte culture methods. This manuscript addresses this challenge by introducing for the first time successful long-term in vitro cultivation of primary human hepatocytes from a range of liver transplantation patients using conditional reprogramming technique. The beauty of this technique is that it is not a permanent immortalization and does not require co-culture with additive cells. The primary human hepatocytes retain proliferative capacity, genetic stability, and hepatocyte-specific functions at early passages. In view of these, we believe that scientists and researchers will benefit from using these highly valuable cell models to study diverse liver diseases.

Epithelium morphogenesis and oviduct development are regulated by significant increase of expression of genes after long-term in vitro primary culture – a microarray assays

Abstract The correct oviductal development and morphogenesis of its epithelium are crucial factors influencing female fertility. Oviduct is involved in maintaining an optimal environment for gametes and preimplantation embryo development; secretory oviductal epithelial cells (OECs) synthesize components of oviductal fluid. Oviductal epithelium also participates in sperm binding and its hyperactivation. For better understanding of the genetic bases that underlay porcine oviductal development, OECs were isolated from porcine oviducts and established long-term primary culture. A microarray approach was utilized to determine the differentially expressed genes during specific time periods. Cells were harvested on day 7, 15 and 30 of in vitro primary culture and their RNA was isolated. Gene expression was analyzed and statistical analysis was performed. 48 differentially expressed genes belonging to “tube morphogenesis”, “tube development”, “morphogenesis of an epithelium”, “morphogenesis of branching structure” and “morphogenesis of branching epithelium” GO BP terms were selected, of which 10 most upregulated include BMP4, ARG1, SLIT2, FGFR1, DAB2, TNC, EPAS1, HHEX, ITGB3 and LOX. The results help to shed light on the porcine oviductal development and its epithelial morphogenesis, and show that after long-term culture the OECs still proliferate and maintain their tube forming properties.

Genes responsible for proliferation, differentiation, and junction adhesion are significantly up-regulated in human ovarian granulosa cells during a long-term primary in vitro culture

The human ovarian granulosa cells (GCs) surround the oocyte and form the proper architecture of the ovarian follicle. The ability of GCs to proliferate and differentiate in the conditions of in vitro culture has been proven. However, there is still a large field for extensive investigation of molecular basics, as well as marker genes, responsible for these processes. This study aimed to find the new marker genes, encoding proteins that regulate human GCs in vitro capability for proliferation and differentiation during long-term primary culture. The human follicular GCs were collected from hyper-stimulated ovarian follicles during IVF procedures and transferred to a long-term in vitro culture. The culture lasted for 30 days, with RNA samples isolated at days 1, 7, 15, 30. Transcriptomic analysis was then performed with the use of Affymetrix microarray. Obtained results were then subjected to bioinformatical evaluation and sorting. After subjecting the datasets to KEGG analysis, three differentially expressed ontology groups “cell differentiation” (GO:0030154), “cell proliferation” (GO:0008283) and “cell–cell junction organization” (GO:0045216) were chosen for further investigation. All three of those ontology groups are involved in human GCs’ in vitro lifespan, proliferation potential, and survival capability. Changes in expression of genes of interest belonging to the chosen GOs were validated with the use of RT-qPCR. In this manuscript, we suggest that VCL, PARVA, FZD2, NCS1, and COL5A1 may be recognized as new markers of GC in vitro differentiation, while KAT2B may be a new marker of their proliferation. Additionally, SKI, GLI2, FERMT2, and CDH2 could also be involved in GC in vitro proliferation and differentiation processes. We demonstrated that, in long-term in vitro culture, GCs exhibit markers that suggest their ability to differentiate into different cells types. Therefore, the higher expression profile of these genes may also be associated with the induction of cellular differentiation processes that take place beyond the long-term primary in vitro culture.

Fatty Acids Related Genes Expression Undergo Substantial Changes in Porcine Oviductal Epithelial Cells During Long-Term Primary Culture

Abstract The process of reproduction requires several factors, leading to successful fertilization of an oocyte by a single spermatozoon. One of them is the complete maturity of an oocyte, which is acquired during long stages of folliculogenesis and oogenesis. Additionally, the oviduct, composed of oviductal epithelial cells (OECs), has a prominent influence on this event through sperm modification and supporting oocyte’s movement towards uterus. OECs were isolated from porcine oviducts. Cells were kept in primary in vitro culture for 30 days. After 24h and on days 7, 15 and 30 cells were harvested, and RNA was isolated. Transcript changes were analyzed using microarrays. Fatty acids biosynthetic process and fatty acids transport ontology groups were selected for analysis and described. Results of this study indicated that majority of genes in both ontology groups were up-regulated on day 7, 15 and 30 of primary in vitro culture. We analyzed genes involved in fatty acids biosynthetic process, including: GGT1, PTGES, INSIG1, SCD, ACSL3, FADS2, FADS1, ACSS2, ALOX5AP, ACADL, SYK, ACACA, HSD17B8, FADS3, OXSM, and transport, including: ABCC2, ACSL4, FABP3, PLA2G3, PPARA, SYK, PPARD, ACACA and P2RX7. Elevated levels of fatty acids in bovine and human oviducts are known to reduce proliferation capacity of OECs and promote inflammatory responses in their microenvironment. Most of measured genes could not be connected to reproductive events. However, the alterations in cellular proliferation, differentiation and genes expression during in vitro long-term culture were significant. Thus, we can treat them as putative markers of changes in OECs physiology.

Extensive and systematic rewiring of histone post-translational modifications in cancer model systems.

Histone post-translational modifications (PTMs) generate a complex combinatorial code that regulates gene expression and nuclear functions, and whose deregulation has been documented in different types of cancers. Therefore, the availability of relevant culture models that can be manipulated and that retain the epigenetic features of the tissue of origin is absolutely crucial for studying the epigenetic mechanisms underlying cancer and testing epigenetic drugs. In this study, we took advantage of quantitative mass spectrometry to comprehensively profile histone PTMs in patient tumor tissues, primary cultures and cell lines from three representative tumor models, breast cancer, glioblastoma and ovarian cancer, revealing an extensive and systematic rewiring of histone marks in cell culture conditions, which includes a decrease of H3K27me2/me3, H3K79me1/me2 and H3K9ac/K14ac, and an increase of H3K36me1/me2. While some changes occur in short-term primary cultures, most of them are instead time-dependent and appear only in long-term cultures. Remarkably, such changes mostly revert in cell line- and primary cell-derived in vivo xenograft models. Taken together, these results support the use of xenografts as the most representative models of in vivo epigenetic processes, suggesting caution when using cultured cells, in particular cell lines and long-term primary cultures, for epigenetic investigations.

P62-ZZ Domain Signaling Inhibition Rescues MM-Induced Epigenetic Repression at the Runx2 promoter and Allows Osteoblast Differentiation of MM Patient Pre-Osteoblasts In Vitro

Multiple myeloma (MM) bone disease is characterized by non-healing lytic bone lesions with highly suppressed or absent osteoblast (OB) function. These lesions persist in the absence of active disease, significantly contributing to patient morbidity and mortality after patients achieve hematologic remission. We previously reported that MM cells induce epigenetic changes at the Runx2 promoter in bone marrow stromal cells (BMSC), which are preOB. We also demonstrated that Gfi1, a transcriptional repressor of the key OB differentiation factor, Runx2, is induced in BMSC by MM. Gfi1 is highly expressed in the MM bone marrow microenvironment and directly binds the Runx2 promoter, recruiting chromatin corepressors such as HDAC1 in preOB to induce epigenetic repression of Runx2 and prevent OB differentiation. Importantly, we recently showed that primary MM patient BMSC cultured in the absence of MM cells have decreased levels of the chromatin activation mark H3K9Ac at the Runx2 gene promoter when compared to healthy donor BMSC, suggesting that MM cells induce persistent epigenetic changes in patient BMSC.We reported that p62 (sequestosome-1) in BMSC is critical for the formation of MM-induced signaling complexes that mediate OB suppression and identified XRK3F2, an inhibitor of the p62 ZZ domain. XRK3F2 blunts MM cell-induced Runx2 suppression and Gfi1 induction in murine preOB in vitro, and induced new bone formation and remodeling in the presence of tumor in vivo. In addition, coculture experiments using human MM cells and murine preOB showed that XRK3F2 both prevents and reverses Gfi1 upregulation. Recently, we demonstrated that XRK3F2 prevents the epigenetic suppression of Runx2 in murine preOB cocultured (48h) with MM cells. Using ChIP-qPCR analysis we found that XRK3F2 prevented MM-induced Gfi1 occupancy at the Runx2 promoter, recruitment of the chromatin corepressor HDAC1, and histone de-acetylation.We now report that XRK3F2 restores OB suppression in persistently suppressed BMSC from MM patients. ChIP analysis of primary MM patient-derived BMSC cultured in the presence of XRK3F2 showed that XRK3F2 rescues H3K9ac levels at the Runx2 promoter. XRK3F2 did not alter H3K9ac levels in healthy donor BMSC or enhance OB differentiation. Importantly, XRK3F2 treatment of long-term primary MM patient BMSC cultures allowed osteogenic differentiation and mineralization, as evidenced by alizarin red staining. Further analysis of these cultures demonstrated that XRK3F2 treatment reduced Gfi1 protein expression.We conclude that XRK3F2 blocks MM-induced signaling in BMSC, resulting in decreased Gfi1 levels, thereby reducing recruitment of Gfi1 and HDAC1 to the Runx2 promoter, and reversing MM-induced epigenetic suppression of Runx2. These results suggest that targeting the p62 ZZ domain may reverse Gfi1 upregulation and rescue MM-induced epigenetic suppression of Runx2 in BMSC, allowing restoration of OB function in patients with MM bone disease. DisclosuresRoodman:Amgen: Consultancy.

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture.

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (>3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for >3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.