An in vitro model for studies of intercellular communication in cultured rat anterior pituitary cells

The formation of intimate associations among different hormone-secreting cells within the rat adenohypophysis may serve as a possible site for physiologic regulation. In this report we describe a high density plating method which enables us to study cell-to-cell interactions within anterior pituitary cell cultures. Trypsin-dispersed pituitary cell suspensions attach rapidly (within 6 hr) and quantitatively (95-97%) to glass or plastic surfaces when plated in medium containing microM calcium concentrations (pH 7.6-7.8). Freshly plated cell suspensions obtained from female pituitary glands contained subpopulations of mammotrophs 49.3%, somatotrophs 30.3%, gonadotrophs 12.6%, corticotrophs 3.4% and thyrotrophs 1.5%. Epithelial cell colonies were formed during a 3-day culture period as the cells flattened and re-established contacts with neighboring cells. Freeze-fracture electron microscopic analysis of these colonies produced morphological evidence for direct intercellular contacts among the hormone-secreting cells. Large areas of tight junctions and small gap junctions were identified on the membranes of the epithelial cells within these colonies. Cells which contained tight junctions usually contained microvilli and morphological signs of active hormone secretion. Small junctional plaques containing tightly packed intramembrane particles were also occasionally found on the membranes of cells which were actively secreting pituitary hormones. The high density plating procedure which is described in this report provides greater opportunity for cell-cell interaction and thus may prove to be a useful model for evaluating the role of intercellular communication within this tissue.     

Analysis of pure and mixed murine mast cell colonies

Mast cells have been proposed to originate from diverse sources, including connective tissues, macrophages, T lymphocytes, and hemopoietic cells. Evidence for a hemopoietic origin of mast cells includes the presence of mast cell precursors in spleen colonies and the presence of mast cells in hemopoietic colonies in culture. Here we report a detailed analysis of mouse spleen mixed hemopoietic colonies containing mast cells. All of the colonies in cultures plated at low cell densities were individually removed for analysis by May-Grunwald-Giemsa staining on day 15 of culture. Examination of five dishes which contained a total of 82 colonies showed 16 pure mast cell colonies and 36 mixed mast cell colonies. Sixteen different combinations of cell types were seen and were not distinguishable from each other in situ. The most diverse type of mixed colony contained macrophages (m), neutrophils (n), eosinophils (e), mast cells (Mast), megakaryocytes (M), erythroid cells (E), and blast cells. The clonal origin of mixed mast cell colonies was established by the replating of single cells obtained from blast cell colonies. Individual cells were removed with a micromanipulator, replated, and allowed to grow for 15 days. Cytospin preparations of 10 such colonies showed diverse combinations of cell lineages which were seen in the different types of mixed mast cell colonies described above. Replating studies of mixed mast cell colonies were carried out and a high incidence of replating was seen. Approximately one half of these colonies formed only mast cell colonies upon replating. Further studies showed that pure mast cell colonies could be serially replated four to five times. The replating efficiency of cells in the primary mast cell colonies varied over a wide range (2.5–44%) with an average replating efficiency of 13%. The data also revealed that cells containing metachromatic granules possess significant proliferative capacity. From these studies of pure and mixed mast cell colonies, we concluded (1) that mast cells are in wide variety of types of mixed colonies and that the in situ identification of mixed colonies is unreliable, (2) that mast cells are derived from pluripotent hemopoietic stem cells, and (3) that mast cells with metachromatic granules can have a high proliferating ability.     

Morphogenesis in cell colonies grown from Convolvulus cell suspensions plated on synthetic media

Filtered cell suspensions of cultured callus tissue derived from the roots of Convolvulus arvensis L. were plated out on synthetic agar nutrient media in petri plates. Cell colonies which formed from the single cells or small cell groups in the suspension showed a considerable range of developmental patterns depending upon the physical and chemical environment to which they were exposed. Variation of the auxin and kinin concentrations and the nature and concentration of the source of reduced N compounds had the most profound effects on colony development. High auxin favored cell enlargement, high kinin favored the development of compact colonies composed of many small cells. Both auxin and kinin were required for cell colony formation. Cell differentiation responses which were observed but not subject to experimental control included formation of starch- and crystal-storing cells, differentiation of tracheary elements, formation of cellular filaments, and development of chlorophyllous tissue. Organ initiation was studied in cell colonies developed directly from plated cell suspensions and in cell colonies subcultured on various nutrient media. Bud initiation was produced repeatedly on media containing NAA at 10^-8 to 10^-6 m combined with kinetin at 10^-6 m . Root initiation was induced infrequently and unpredictably. Once roots had been formed from cell colonies derived from cell suspensions, the roots could be subcultured and induced to form buds; these in turn grew into whole plants. Subculture of young cell colonies to media containing different combinations of growth substances made possible a study of the effects of auxin and kinin on organization of primordia by the cell colonies. By following marked single cells plated on synthetic media, it was possible to produce single-cell clones which under proper nutrient conditions were induced to form buds. The value of the combined techniques of cell suspension culture and cell plating for the study of the physical and chemical factors influencing cell differentiation and organized development are pointed out.     

Extrinsic factors derived from mouse embryonal carcinoma cell lines maintain pluripotency of mouse embryonic stem cells through a novel signal pathway

Embryonic carcinoma (EC) cells, which are malignant stem cells of teratocarcinoma, have numerous morphological and biochemical properties in common with pluripotent stem cells such as embryonic stem (ES) cells. However, three EC cell lines (F9, P19 and PCC3) show different developmental potential and self‐renewal capacity from those of ES cells. All three EC cell lines maintain self‐renewal capacity in serum containing medium without Leukemia Inhibitory factor (LIF) or feeder layer, and show limited differentiation capacity into restricted lineage and cell types. To reveal the underlying mechanism of these characteristics, we took the approach of characterizing extrinsic factors derived from EC cells on the self‐renewal capacity and pluripotency of mouse ES cells. Here we demonstrate that EC cell lines F9 and P19 produce factor(s) maintaining the undifferentiated state of mouse ES cells via an unidentified signal pathway, while P19 and PCC3 cells produce self‐renewal factors of ES cells other than LIF that were able to activate the STAT3 signal; however, inhibition of STAT3 activation with Janus kinase inhibitor shows only partial impairment on the maintenance of the undifferentiated state of ES cells. Thus, these factors present in EC cells‐derived conditioned medium may be responsible for the self‐renewal capacity of EC and ES cells independently of LIF signaling.     

Cryopreservation and subsequent viability of human bone marrow in hematologic malignancies: Comparison of two different methods of cell reconstitution

Bone marrow cells collected from patients with hematologic malignancies were cryopreserved using DMSO as a cryoprotective agent. The growth kinetics of hemopoietic stem cells frozen to −196 °C was monitored immediately after thawing by the semisolid agar CFU-C assay and two different methods of cell reconstitution were compared. In the first procedure, thawed cells were plated after the removal of DMSO by washing the cell suspension; in the second, cell suspensions were cultured after a simple 1:1 dilution of DMSO with medium. The numbers of CFU-C per 2 × 10⁵ cells plated was higher by washing out the DMSO in all the groups studied. However, the absolute numbers of CFU-C contained in the whole ampoules after the freezing procedures was approximately the same using both methods. It is concluded that washing the cells only apparently yielded a better cloning efficiency, suggesting that such a procedure led to a higher mature nucleated cell loss with the consequence of a CFU-C concentration. This trend seems particularly evident in cells from the AML and CML patients.     

Determination of numbers of osteoprogenitors present in isolated fetal rat calvaria cells in vitro

When maintained in long-term cell culture in the presence of ascorbic acid and organic phosphate, single cell suspensions isolated from fetal rat calvaria form discrete, three-dimensional bone nodules. We have used limiting dilution analysis in microtiter wells to determine the number of osteoprogenitor cells expressing the capacity to form bone in the isolated mixed population, to examine the possibility of cooperativity among cell types in bone nodule formation, and to determine the effects of dexamethasone on osteoprogenitor cells. Cells plated at very low densities and screened for the presence or absence of bone nodules revealed a linear relationship (r = -00.997) between the number of cells plated and the number of bone nodules formed. The complete limiting dilution analyses showed that 1 of every 335 plated cells (0.30% of the cell population) has the capacity to form a bone nodule under standard culture conditions and when the actual numbers of nodules were quantitated from the same plated cell populations the ratio of nodules formed to plated cells was similar. Comparison of data from 13 different isolates of cells in which cells were plated into 35-mm dishes and number of nodules were determined indicated a mean +/- 95% confidence interval of one nodule for every 301 +/- 61 plated cells, consistent with the data obtained from the limiting dilution experiments. Dexamethasone increased the number of bone-forming cells to 1 in 225 cells, in contrast to 1 in 340 cells in the same population grown without added dexamethasone. The results suggest that approximately 0.30% of the cells in isolated rat calvaria populations are osteoprogenitor cells, that one osteoprogenitor cell gives rise to one bone nodule, that cooperativity between different cells in vitro is not necessary for bone formation, and that dexamethasone stimulates the expression of osteoprogenitor cells.     

Proliferative kinetics and differentiation of murine blast cell colonies in culture: Evidence for variable G0 periods and constant doubling rates of early pluripotent hemopoietic progenitors

Several investigators have described hemopoietic colonies expressing multilineage differentiation in culture. We recently identified a class of murine hemopoletic progenitors which form blast cell colonies with very high replating efficiencies. In order to clarify further the relationship between progenitors for blast cell colonies and progenitors for the multilineage hemopoietic colonies in culture, we carried out analyses of kinetic and differentiation properties of murine blast cell colonies. Serial observations of the development of blast cell colonies into multilineage (and single lineage) colonies in cultures of spleen cells obtained from 5-fluorouracil (5-FU)-treated mice confirmed the transitional nature of the murine blast cell colonies. The data also suggested that the early pluripotent progenitors are in G₀ for variable periods, and that when triggered into cell cycle, they proliferate at relatively constant doubling rates during the early stages of differentiation. The notion that some of the pluripotent progenitors are in G₀ was also supported by long-term thymidine suicide studies in which spleen cells were exposed to ³H-thymidine with high specific activity for 5 days in culture, washed, and assayed for surviving progenitors. Comparison of replating abilities of day-7 and day-16 blast cell colonies from normal as well as 5-FU-treated mice indicated that some of the day-7 blast cell colonies are derived from maturer populations of progenitors which are sensitive to 5-FU. In contrast, progenitors for the day-16 blast cell colonies are dormant in cell cycle and were not affected by 5-FU treatment. Previously we reported that progenitors for day-16 blast cell colonies have a significant capacity for self-renewal. These observations suggest the hypothesis that the capability for self-renewal is accompanied by long periods of G₀, and that once commitment to differentiation takes place, then active cell division occurs.     

Similarities in long-term cultures of blood and bone marrow from patients with acute myelogenous leukemia

Dexter-type long-term cultures (LTC) were initiated with peripheral blood (PB) and/or bone marrow cells from 11 patients with acute myelogenous leukemia (AML), and 2 with myelodysplastic syndrome in blastic transformation. Marrow and PB cells from normal subjects served as controls. Assessment of nucleated cells and clonogenic progenitors in the adherent and nonadherent fractions of LTC revealed active hemopoiesis for greater than 5 wks in 4 of 8 cultures of AML blood, and 4 of 7 of AML marrow. The morphology and kinetics of nucleated cells and progenitors with putative normal (granulocyte-macrophage colony-forming units or CFU-gm), and abnormal (blast) phenotype in LTC from AML blood were similar to those from AML marrow, and adherent cells positive for collagen I and III and vimentin were found in both types of LTC. Growth of CFU-gm colonies ceased by wk 5-8 in AML cultures, significantly earlier than in LTC of normal marrow cells (survival of greater than 10 wks), which may indicate derivation of the CFU-gm from a transformed clone or deficiency of stromal function in the leukemic state. In most AML blood and AML marrow LTCs, growth of abnormal (blast) colonies continued until wk 4-6. This study demonstrates certain similarities of morphology and function between LTC of AML blood and AML marrow cells. LTC may provide a useful model for further analysis of circulating primitive hemopoietic progenitor cells in leukemic states.     

COLONY FORMATION IN AGAR: IN VITRO ASSAY FOR HAEMOPOIETIC STEM CELLS

Using a method in which embryo fibroblasts were used as feeder layers, the colony forming capacity in agar of a variety of mouse haemopoietic suspensions was compared with their CFU s content. A striking parallelism between the results of the two assays was found. In addition, under certain conditions higher numbers of CFU s could be retrieved from 5-day-old agar colonies than were originally plated, indicating that the CFC a (Colony Forming Cell agar) may fulfil the requirements of pluripotency as well as of self-renewal, both prerequisites for any haemopoietic stem cell candidate. Although our data by no means provide direct proof that the CFC s and the CFC a are identical, they certainly support such a concept. the contradictory findings by others that CFU s and CFU c (Colony Forming Unit culture) can be separated on a velocity gradient is attributed to different culture conditions, in other words, that their CFU cè are not identical with our CFU a .
Our findings also indicate that for mouse cells our soft agar colony assay meets the criteria of a quantitative assay for haemopoietic stem cells and that extension of this technique to bone marrow of primates including humans seems to be justified.     

Pluripotential hematopoietic stem cells in post-5-fluorouracil murine bone marrow express the Thy-1 antigen

Expression of the Thy-1 alloantigen by hematopoietic stem and progenitor cells in post-5-fluorouracil (5-FU) murine bone marrow was investigated. FACS analysis of BDF1 bone marrow stained for Thy-1.2 with a triple-layer amplified labeling technique demonstrated that 35% of the total bone marrow population expressed Thy-1.2 (Thy-1.2+). Two distinct size subpopulations were observed in post-5-FU BDF1 marrow. Thy-1.2+ cells were present in both the large and the small subpopulations. FACS-separated bone marrow cells were also plated in methylcellulose cultures. Ninety percent of all colony-forming cells surviving in vivo administration of 5-FU were Thy-1.2+. Replating of primary hemopoietic colonies and morphologic examination of primary and secondary colonies demonstrated that the most primitive stem cells including "stem" (S) cells were Thy-1.2+. These cells (Thy-1.2+) were capable of self-renewal in vitro and exhibited multiple differentiation potentials in comparison to Thy-1.2-cells, which lacked significant self-renewal capability and were mono- or bipotent progenitor cells. Separation of Thy-1.2+ cells into large or small Thy-1.2+ subpopulations showed that only the large Thy-1.2+ colony-forming cells possessed significant self-renewal capacity. Treatment of BDF1 bone marrow with anti-Thy-1.2 plus complement reduced primary colony formation by 67% and eliminated those colony-forming cells which had extensive self-renewal properties. In the presence of PWMSCM, depletion and reconstitution of T lymphocytes had no effect on primary or secondary colony formation. These data demonstrate that Thy-1 is present on primitive hematopoietic stem cells in post-5-FU bone marrow. In addition, they show that the murine S cell is Thy-1+.     

Targeting miR‐193a‐AML1‐ETO‐β‐catenin axis by melatonin suppresses the self‐renewal of leukaemia stem cells in leukaemia with t (8;21) translocation

AML1‐ETO, the most common fusion oncoprotein by t (8;21) in acute myeloid leukaemia (AML), enhances hematopoietic self‐renewal and leukemogenesis. However, currently no specific therapies have been reported for t (8;21) AML patients as AML1‐ETO is still intractable as a pharmacological target. For this purpose, leukaemia cells and AML1‐ETO‐induced murine leukaemia model were used to investigate the degradation of AML1‐ETO by melatonin (MLT), synthesized and secreted by the pineal gland. MLT remarkedly decreased AML1‐ETO protein in leukemic cells. Meanwhile, MLT induced apoptosis, decreased proliferation and reduced colony formation. Furthermore, MLT reduced the expansion of human leukemic cells and extended the overall survival in U937T‐AML1‐ETO‐xenografted NSG mice. Most importantly, MLT reduced the infiltration of leukaemia blasts, decreased the frequency of leukaemia stem cells (LSCs) and prolonged the overall survival in AML1‐ETO‐induced murine leukaemia. Mechanistically, MLT increased the expression of miR‐193a, which inhibited AML1‐ETO expression via targeting its putative binding sites. Furthermore, MLT decreased the expression of β‐catenin, which is required for the self‐renewal of LSC and is the downstream of AML1‐ETO. Thus, MLT presents anti‐self‐renewal of LSC through miR‐193a‐AML1‐ETO‐β‐catenin axis. In conclusion, MLT might be a potential treatment for t (8;21) leukaemia by targeting AML1‐ETO oncoprotein.     

Recombinant murine granulocyte-macrophage (GM) colony-stimulating factor supports formation of GM and multipotential blast cell colonies in culture: comparison with the effects of interleukin-3

We studied the effects of murine recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) on murine hemopoiesis in methylcellulose culture. The GM-CSF was purified from cultures of Saccharomyces cerevisiae transfected with a cloned murine GM-CSF cDNA. In cultures of spleen cells from normal mice, only granulocyte-macrophage (GM) colonies were supported by GM-CSF. Blast cell colonies were the predominant type in cultures of spleen cells from 5-fluorouracil (5-FU)-treated mice. Dose-response studies revealed that maximal GM and blast cell colony formation is achieved with 100 U/ml GM-CSF. Blast cell colonies revealed variable but high replating efficiencies, and the secondary colonies included multilineage colonies. Serial replating of washed blast cell colonies in cultures with GM-CSF provided evidence for the direct effects of GM-CSF on the proliferation of multipotential blast cells. A combination of GM-CSF and interleukin-3 (IL-3) did not increase the number of blast cell colonies over the level supported by IL-3. This observation indicates that the progenitors for blast cell colonies that responded to GM-CSF are a subpopulation of multipotential progenitors that are supported by IL-3. Cytological studies of colonies derived from GM-CSF and/or IL-3 suggest that the eosinophilopoietic ability of murine GM-CSF is less than that of IL-3.     

Murine B-cell stimulatory factor-1 (BSF-1)/interleukin-4 (IL-4) is a multilineage colony-stimulating factor that acts directly on primitive hemopoietic progenitors

Interleukin-4 (IL-4), which was originally identified as a B-cell growth factor, has been shown to produce diverse effects on hemopoietic progenitors. The present study investigated the effects of purified recombinant murine IL-4 on early hemopoetic progenitors in methylcellulose culture. IL-4 supported the formation of blast cell colonies and small granulocyte/macrophage (GM) colonies in cultures of marrow and spleen cells of normal mice as well as spleen cells of mice treated with 150 mg/kg 5-fluorouracil (5-FU) 4 days earlier. When the blast cell colonies were individually picked and replated in cultures containing WEHI-3 conditioned medium and erythropoietin (Ep), a variety of colonies were seen, including mixed erythroid colonies, indicating the multipotent nature of the blast cell colonies supported by IL-4. To test whether or not IL-4 affects multipotent progenitors directly, we replated pooled blast cells in cultures under varying conditions. In the presence of Ep, both IL-3 and IL-4 supported a similar number of granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colonies. However, the number of GM colonies supported by IL-4 was significantly smaller than that supported by IL-3. When colony-supporting abilities of IL-4 and IL-3 were compared using day-4 post-5-FU spleen and day-2 post-5-FU marrow cells, IL-4 supported the formation of fewer blast cell colonies than did IL-3. IL-4 and IL-6 revealed synergy in support of colony formation from day 2 post-5-FU marrow cells. These results indicate that murine IL-4 is another direct-acting multilineage colony-stimulating factor (multi-CSF), similar to IL-3, that acts on primitive hemopoietic progenitors.     

Smurf2 regulates hematopoietic stem cell self‐renewal and aging

The age‐dependent decline in the self‐renewal capacity of stem cells plays a critical role in aging, but the precise mechanisms underlying this decline are not well understood. By limiting proliferative capacity, senescence is thought to play an important role in age‐dependent decline of stem cell self‐renewal, although direct evidence supporting this hypothesis is largely lacking. We have previously identified the E3 ubiquitin ligase Smurf2 as a critical regulator of senescence. In this study, we found that mice deficient in Smurf2 had an expanded hematopoietic stem cell (HSC) compartment in bone marrow under normal homeostatic conditions, and this expansion was associated with enhanced proliferation and reduced quiescence of HSCs. Surprisingly, increased cycling and reduced quiescence of HSCs in Smurf2‐deficient mice did not lead to premature exhaustion of stem cells. Instead, HSCs in aged Smurf2‐deficient mice had a significantly better repopulating capacity than aged wild‐type HSCs, suggesting that decline in HSC function with age is Smurf2 dependent. Furthermore, Smurf2‐deficient HSCs exhibited elevated long‐term self‐renewal capacity and diminished exhaustion in serial transplantation. As we found that the expression of Smurf2 was increased with age and in response to regenerative stress during serial transplantation, our findings suggest that Smurf2 plays an important role in regulating HSC self‐renewal and aging.     

MUC1‐C drives myeloid leukaemogenesis and resistance to treatment by a survivin‐mediated mechanism

Acute myeloid leukaemia (AML) is an aggressive haematological malignancy with an unmet need for improved therapies. Responses to standard cytotoxic therapy in AML are often transient because of the emergence of chemotherapy‐resistant disease. The MUC1‐C oncoprotein governs critical pathways of tumorigenesis, including self‐renewal and survival, and is aberrantly expressed in AML blasts and leukaemia stem cells (LSCs). However, a role for MUC1‐C in linking leukaemogenesis and resistance to treatment has not been described. In this study, we demonstrate that MUC1‐C overexpression is associated with increased leukaemia initiating capacity in an NSG mouse model. In concert with those results, MUC1‐C silencing in multiple AML cell lines significantly reduced the establishment of AML in vivo. In addition, targeting MUC1‐C with silencing or pharmacologic inhibition with GO‐203 led to a decrease in active β‐catenin levels and, in‐turn, down‐regulation of survivin, a critical mediator of leukaemia cell survival. Targeting MUC1‐C was also associated with increased sensitivity of AML cells to Cytarabine (Ara‐C) treatment by a survivin‐dependent mechanism. Notably, low MUC1 and survivin gene expression were associated with better clinical outcomes in patients with AML. These findings emphasize the importance of MUC1‐C to myeloid leukaemogenesis and resistance to treatment by driving survivin expression. Our findings also highlight the potential translational relevance of combining GO‐203 with Ara‐C for the treatment of patients with AML.     

Clonal nature of murine hemopoietic blast cell colonies

Murine hemopoietic blast cell colonies obtained from spleen cells of 5-fluorouracil (5-FU)-treated mice give rise to many multilineage colonies including granulocyte - erythrocyte - macrophage - megakaryocyte (GEMM) colonies in secondary cultures. Progenitor cells for blast cell colonies are considered to be more primitive than colony forming units (CFU)-GEMM. To determine whether they are clonal, we examined the phosphoglycerate kinase-1 (PGK-1) isozyme type of colonies originally grown from spleen cells of 5-FU-treated mice which had PGK-1 isozyme mosaicism. PGK assays of whole secondary colonies derived from one blast cell colony showed that they were either of type A or type B but not both. These results suggest that murine hemopoietic blast cell colonies are clonal.     

CFU-F from dog marrow: a colony assay and its significance

A colony assay method is described for studying dog fibroblast colony development in marrow cells derived from resected ribs. The assay showed an increased number of fibroblast colony forming units (CFU-F) in cell suspensions prepared from resected ribs compared to cell suspensions prepared from bone marrow aspirates or from peripheral blood. A linear relationship between the number of cells plated and the number of fibroblastoid colonies was demonstrated in each case. The proportion of phagocytic cells was lower in cultures prepared from resected ribs than in those prepared from bone marrow aspirates. Staining for acid phosphatase and with sudan black showed differences between phagocytic cells and non-phagocytic fibroblasts. When left in plastic dishes for 2 hrs, 81% +/- 10% of the CFU-F adhered to the plastic dishes. Velocity sedimentation separation showed a modal sedimentation rate of 6.49 mm/h.     

抑制MTM1表达促进胚胎干细胞分化

应用随机RNAi文库,筛选了与胚胎干细胞自我更新和分化调控相关基因,发现了多个阳性候选基因,对其中的1个阳性候选基因肌管素1（myotubularin, MTM1）基因进行了深入研究．MTM1是属于蛋白酪氨酸磷酸酶（PTPase）蛋白家族的蛋白,其基因突变导致肌管性肌病．MTM1在胚胎干细胞中的功能到目前为止还不清楚．研究证实,MTM1在小鼠胚胎干细胞系CCE和R1均有表达．应用RNA干扰及集落形成实验证明,MTM1表达抑制后,处于自我更新状况胚胎干细胞集落的比例显著增加,提示MTM1在胚胎干细胞自我更新和分化的调控中起了重要的作用．     

Spontaneous cell hybridization of somatic cells present in sperm suspensions

Electron microscopic evidence suggests that sperm can be spontaneously incorporated by cultured cells but cytogenetic and biochemical evidence indicate that sperm do not introduce new genes into such cells with detectable frequency. Sperm suspensions from mouse or Chinese hamster epididymis or human semen were added to cultures of RAG, a mouse cell line which dies in HAT medium because of HPRT deficiency. In EMs, sperm appeared to be readily phagocytized and degraded by the cells. When sperm-treated cultures were transferred to HAT medium resistant clones arose at a frequency of about 10⁻⁶, or at least 25× the reversion rate of RAG. Most HAT-resistant clones had HPRT activity which migrated electrophoretically like HPRT of the sperm donor species, though one was apparently a spontaneous RAG revertant. Most HAT-resistant clones had some chromosomes of the sperm donor species. In human sperm× RAG clones, the array of human chromosomes suggested that the human parent had been diploid rather than haploid; some cells contained both homologues of a polymorphic pair and some contained both X and Y. Furthermore, some sperm suspensions plated alone into flasks generated colonies, thus revealing the presence of low numbers of viable somatic cells. Presence of contaminating somatic cells in a sperm suspension was correlated with ability to induce HAT-resistant colonies when the suspension was added to RAG cells. Taken together, the data suggest that correction of the HPRT deficiency of RAG by sperm suspensions occurs at very low frequency and is probably due to efficient spontaneous fusion of low numbers of contaminating somatic cells with RAG cells.