Exploiting Human CD34+ Stem Cell–conditioned Medium for Tissue Repair

Despite the progress in our understanding of genes essential for stem cell regulation and development, little is known about the factors secreted by stem cells and their effect on tissue regeneration. In particular, the factors secreted by human CD34⁺ cells remain to be elucidated. We have approached this challenge by performing a cytokine/growth factor microarray analysis of secreted soluble factors in medium conditioned by adherent human CD34⁺ cells. Thirty-two abundantly secreted factors have been identified, all of which are associated with cell proliferation, survival, tissue repair, and wound healing. The cultured CD34⁺ cells expressed known stem cell genes such as Nanog, Oct4, Sox2, c-kit, and HoxB4. The conditioned medium containing the secreted factors prevented cell death in liver cells exposed to liver toxin in vitro via inhibition of the caspase-3 signaling pathway. More importantly, in vivo studies using animal models of liver damage demonstrated that injection of the conditioned medium could repair damaged liver tissue (significant reduction in the necroinflammatory activity), as well as enable the animals to survive. Thus, we demonstrate that medium conditioned by human CD34⁺ cells has the potential for therapeutic repair of damaged tissue in vivo.     

ApoE regulates hematopoietic stem cell proliferation, monocytosis, and monocyte accumulation in atherosclerotic lesions in mice

Leukocytosis is associated with increased cardiovascular disease risk in humans and develops in hypercholesterolemic atherosclerotic animal models. Leukocytosis is associated with the proliferation of hematopoietic stem and multipotential progenitor cells (HSPCs) in mice with deficiencies of the cholesterol efflux–promoting ABC transporters ABCA1 and ABCG1 in BM cells. Here, we have determined the role of endogenous apolipoprotein-mediated cholesterol efflux pathways in these processes. In Apoe^–/– mice fed a chow or Western-type diet, monocytosis and neutrophilia developed in association with the proliferation and expansion of HSPCs in the BM. In contrast, Apoa1^–/– mice showed no monocytosis compared with controls. ApoE was found on the surface of HSPCs, in a proteoglycan-bound pool, where it acted in an ABCA1- and ABCG1-dependent fashion to decrease cell proliferation. Accordingly, competitive BM transplantation experiments showed that ApoE acted cell autonomously to control HSPC proliferation, monocytosis, neutrophilia, and monocyte accumulation in atherosclerotic lesions. Infusion of reconstituted HDL and LXR activator treatment each reduced HSPC proliferation and monocytosis in Apoe^–/– mice. These studies suggest a specific role for proteoglycan-bound ApoE at the surface of HSPCs to promote cholesterol efflux via ABCA1/ABCG1 and decrease cell proliferation, monocytosis, and atherosclerosis. Although endogenous apoA-I was ineffective, pharmacologic approaches to increasing cholesterol efflux suppressed stem cell proliferative responses.     

Context aware spatio-temporal cell tracking in densely packed multilayer tissues

Modern live imaging technique enables us to observe the internal part of a tissue over time by generating serial optical images containing spatio-temporal slices of hundreds of tightly packed cells. Automated tracking of plant and animal cells from such time lapse live-imaging datasets of a developing multicellular tissue is required for quantitative, high throughput analysis of cell division, migration and cell growth. In this paper, we present a novel cell tracking method that exploits the tight spatial topology of neighboring cells in a multicellular field as contextual information and combines it with physical features of individual cells for generating reliable cell lineages. The 2D image slices of multicellular tissues are modeled as a conditional random field and pairwise cell to cell similarities are obtained by estimating marginal probability distributions through loopy belief propagation on this CRF. These similarity scores are further used in a spatio-temporal graph labeling problem to obtain the optimal and feasible set of correspondences between individual cell slices across the 4D image dataset. We present results on (3D + t) confocal image stacks of Arabidopsis shoot meristem and show that the method is capable of handling many visual analysis challenges associated with such cell tracking problems, viz. poor feature quality of individual cells, low SNR in parts of images, variable number of cells across slices and cell division detection.     

Electromagnetic field-assisted cell-laden 3D printed poloxamer-407 hydrogel for enhanced osteogenesis

3D bioprinted hydrogel has gained enormous attention, especially in tissue engineering, owing to its attractive structure and excellent biocompatibility. In this study, we demonstrated that 3D bioprinted cell-laden ‘thermoresponsive’ poloxamer-407 (P407) gels have the potential to stimulate osteogenic differentiation of apical papilla stem cells (SCAPs) under the influence of low voltage–frequency (5 V–1 Hz, 0.62 mT) electromagnetic fields (EMFs). SCAPs were initially used for cell-laden 3D printing to biomimic the apical papilla of human teeth. The developed hydrogel exhibited higher mechanical strength as well as good printability, showing high-quality micro-architecture. Moreover, the as-printed hydrogels (5 mm × 5 mm) were loaded with plasminogen activator inhibitor-1 (PAI-1) for testing the combined effect of PAI-1 and EMFs on SCAP differentiation. Interestingly, the 3D hydrogels showed improved viability and differentiation of SCAPs under EMFs'' influence as examined by live/dead assay and alizarin Red-S staining, respectively. Therefore, our results confirmed that P407 hydrogels are non-toxic for encapsulation of SCAPs, yielding high cell viability and accelerate the cell migration potential. The 3D hydrogels with PAI-1 exhibited high mRNA expression levels for osteogenic/odontogenic gene markers (ALP, Col-1, DSPP, and DMP-1) vis-à-vis control after 14 days of in vitro culture. Our findings suggest that 3D bioprinted P407 hydrogels are biocompatible for SCAP encapsulation, and the applied low voltage–frequency EMFs could effectively improve dental tissue regeneration, particularly for oral applications.

3D bioprinted ‘mini-tooth’ respond to low voltage-frequency electromagetic fields (EMFs) stimulation and promote differentiation of apical papilla stem cells (SCAPs).     

Agonism of Wnt–β‐catenin signalling promotes mesenchymal stem cell (MSC) expansion

Promoting mesenchymal stem cell (MSC) proliferation has numerous applications in stem cell therapies, particularly in the area of regenerative medicine. In order for cell‐based regenerative approaches to be realized, MSC proliferation must be achieved in a controlled manner without compromising stem cell differentiation capacities. Here we demonstrate that 6‐bromoindirubin‐3′‐oxime (BIO) increases MSC β‐catenin activity 106‐fold and stem cell‐associated gene expression ~33‐fold, respectively, over untreated controls. Subsequently, BIO treatment increases MSC populations 1.8‐fold in typical 2D culture conditions, as well as 1.3‐fold when encapsulated within hydrogels compared to untreated cells. Furthermore, we demonstrate that BIO treatment does not reduce MSC multipotency where MSCs maintain their ability to differentiate into osteoblasts, chondrocytes and adipocytes using standard conditions. Taken together, our results demonstrate BIO's potential utility as a proliferative agent for cell transplantation and tissue regeneration. Copyright © 2013 John Wiley & Sons, Ltd.     

Harnessing endogenous stem/progenitor cells for tendon regeneration

Current stem cell–based strategies for tissue regeneration involve ex vivo manipulation of these cells to confer features of the desired progenitor population. Recently, the concept that endogenous stem/progenitor cells could be used for regenerating tissues has emerged as a promising approach that potentially overcomes the obstacles related to cell transplantation. Here we applied this strategy for the regeneration of injured tendons in a rat model. First, we identified a rare fraction of tendon cells that was positive for the known tendon stem cell marker CD146 and exhibited clonogenic capacity, as well as multilineage differentiation ability. These tendon-resident CD146⁺ stem/progenitor cells were selectively enriched by connective tissue growth factor delivery (CTGF delivery) in the early phase of tendon healing, followed by tenogenic differentiation in the later phase. The time-controlled proliferation and differentiation of CD146⁺ stem/progenitor cells by CTGF delivery successfully led to tendon regeneration with densely aligned collagen fibers, normal level of cellularity, and functional restoration. Using siRNA knockdown to evaluate factors involved in tendon generation, we demonstrated that the FAK/ERK1/2 signaling pathway regulates CTGF-induced proliferation and differentiation of CD146⁺ stem/progenitor cells. Together, our findings support the use of endogenous stem/progenitor cells as a strategy for tendon regeneration without cell transplantation and suggest this approach warrants exploration in other tissues.     

Use of bone marrow derived stem cells in a fracture non-union

This is an attempt of using in vitro cultured mesenchymal stem cells (MSCs) from bone marrow in joining of a fracture non-union. Bone marrow cells were obtained and differentially centrifuged for MSCs that were grown in vitro in mesenchymal stem cell basal medium aseptically, for 10 d. The cell mass was injected around the fracture non-union. Healthy conditions of development of tissue regeneration at the trauma site and due bone joining were recorded. It is concluded that in vitro cultured MSCs had a blithesome effect on the fracture non-union.     

Long-lived intestinal tuft cells serve as colon cancer–initiating cells

Doublecortin-like kinase 1 protein (DCLK1) is a gastrointestinal tuft cell marker that has been proposed to identify quiescent and tumor growth–sustaining stem cells. DCLK1⁺ tuft cells are increased in inflammation-induced carcinogenesis; however, the role of these cells within the gastrointestinal epithelium and their potential as cancer-initiating cells are poorly understood. Here, using a BAC-CreERT–dependent genetic lineage–tracing strategy, we determined that a subpopulation of DCLK1⁺ cells is extremely long lived and possesses rare stem cell abilities. Moreover, genetic ablation of Dclk1 revealed that DCLK1⁺ tuft cells contribute to recovery following intestinal and colonic injury. Surprisingly, conditional knockdown of the Wnt regulator APC in DCLK1⁺ cells was not sufficient to drive colonic carcinogenesis under normal conditions; however, dextran sodium sulfate–induced (DSS-induced) colitis promoted the development of poorly differentiated colonic adenocarcinoma in mice lacking APC in DCLK1⁺ cells. Importantly, colonic tumor formation occurred even when colitis onset was delayed for up to 3 months after induced APC loss in DCLK1⁺ cells. Thus, our data define an intestinal DCLK1⁺ tuft cell population that is long lived, quiescent, and important for intestinal homeostasis and regeneration. Long-lived DCLK1⁺ cells maintain quiescence even following oncogenic mutation, but are activated by tissue injury and can serve to initiate colon cancer.     

人胎儿肝干细胞在聚酸酐-氨基葡聚糖三维载体内的生长活性

目的:观察聚酸酐-氨基葡聚糖三维载体材料对胎儿肝干细胞黏附及增殖的影响.方法:采用改进的两步胶原酶灌注消化法加percoll液不连续密度梯度离心的方法,分离胎儿肝干细胞.选取胎儿肝干细胞的第3代细胞,种植于聚酸酐-氨基葡聚糖三维载体材料上.倒置显微镜下观察细胞的黏附和生长状况;计算细胞贴壁率、细胞增殖活力、计数细胞数.取细胞-载体进行组织学切片,苏木精-伊红染色光镜下观察细胞在载体中生长情况.在细胞培养第7天进行免疫荧光化学染色和流式细胞仪检测标志物表达.结果:聚酸酐-氨基葡聚糖三维载体能促进肝干细胞在材料内黏附并保持其在机体内的形态.载体材料内的肝干细胞功能活跃,在材料表面和三维空间内部培养的肝干细胞均能持续增殖.经过连续10 d共同培养,聚酸酐-氨基葡聚糖三维载体对干细胞无毒性,人胎儿肝干细胞可以很好的贴附于聚酸酐-氨基葡聚糖三维载体支架上,细胞增殖活力良好,标志物持续表达,培养7 d得到的细胞数量增多19.7%.结论:聚酸酐-氨基葡聚糖三维载体能促进肝干细胞的增殖,有可能作为肝干细胞的载体应用于肝脏组织工程.     

“The Good into the Pot, the Bad into the Crop!”—A New Technology to Free Stem Cells from Feeder Cells

A variety of embryonic and adult stem cell lines require an intial co-culturing with feeder cells for non-differentiated growth, self renewal and maintenance of pluripotency. However for many downstream ES cell applications the feeder cells have to be considered contaminations that might interfere not just with the analysis of experimental data but also with clinical application and tissue engineering approaches. Here we introduce a novel technique that allows for the selection of pure feeder-freed stem cells, following stem cell proliferation on feeder cell layers. Complete and reproducible separation of feeder and embryonic stem cells was accomplished by adaptation of an automated cell selection system that resulted in the aspiration of distinct cell colonies or fraction of colonies according to predefined physical parameters. Analyzing neuronal differentiation we demonstrated feeder-freed stem cells to exhibit differentiation potentials comparable to embryonic stem cells differentiated under standard conditions. However, embryoid body growth as well as differentiation of stem cells into cardiomyocytes was significantly enhanced in feeder-freed cells, indicating a feeder cell dependent modulation of lineage differentiation during early embryoid body development. These findings underline the necessity to separate stem and feeder cells before the initiation of in vitro differentiation. The complete separation of stem and feeder cells by this new technology results in pure stem cell populations for translational approaches. Furthermore, a more detailed analysis of the effect of feeder cells on stem cell differentiation is now possible, that might facilitate the identification and development of new optimized human or genetically modified feeder cell lines.     

In Vivo Myogenic Potential of Human CD133+ Muscle-derived Stem Cells: A Quantitative Study

In recent years, numerous reports have identified in mouse different sources of myogenic cells distinct from satellite cells that exhibited a variable myogenic potential in vivo. Myogenic stem cells have also been described in humans, although their regenerative potential has rarely been quantified. In this study, we have investigated the myogenic potential of human muscle–derived cells based on the expression of the stem cell marker CD133 as compared to bona fide satellite cells already used in clinical trials. The efficiency of these cells to participate in muscle regeneration and contribute to the renewal of the satellite cell pool, when injected intramuscularly, has been evaluated in the Rag2^−/− γC^−/− C5^−/− mouse in which muscle degeneration is induced by cryoinjury. We demonstrate that human muscle–derived CD133⁺ cells showed a much greater regenerative capacity when compared to human myoblasts. The number of fibers expressing human proteins and the number of human cells in a satellite cell position are all dramatically increased when compared to those observed after injection of human myoblasts. In addition, CD133⁺/CD34⁺ cells exhibited a better dispersion in the host muscle when compared to human myoblasts. We propose that muscle-derived CD133⁺ cells could be an attractive candidate for cellular therapy.     

Hypoxia Preconditioned Mesenchymal Stem Cells Improve Vascular and Skeletal Muscle Fiber Regeneration After Ischemia Through a Wnt4-dependent Pathway

Mesenchymal stem cells (MSC) are multipotent postnatal stem cells, involved in the treatment of ischemic vascular diseases. We investigate the ability of MSC, exposed to short-term hypoxic conditions, to participate in vascular and tissue regeneration in an in vivo model of hindlimb ischemia. Transplantation of hypoxic preconditioned murine MSC (HypMSC) enhanced skeletal muscle regeneration at day 7, improved blood flow and vascular formation compared to injected nonpreconditioned MSC (NormMSC). These observed effects were correlated with an increase in HypMSC engraftment and a putative role in necrotic skeletal muscle fiber clearance. Moreover, HypMSC transplantation resulted in a large increase in Wnt4 (wingless-related MMTV integration site 4) expression and we demonstrate its functional significance on MSC proliferation and migration, endothelial cell (EC) migration, as well as myoblast differentiation. Furthermore, suppression of Wnt4 expression in HypMSC, abrogated the hypoxia-induced vascular regenerative properties of these cells in the mouse hindlimb ischemia model. Our data suggest that hypoxic preconditioning plays a critical role in the functional capabilities of MSC, shifting MSC location in situ to enhance ischemic tissue recovery, facilitating vascular cell mobilization, and skeletal muscle fiber regeneration via a paracrine Wnt-dependent mechanism.     

A critical role for Apc in hematopoietic stem and progenitor cell survival

The adenomatous polyposis coli (Apc) tumor suppressor is involved in the initiation and progression of colorectal cancer via regulation of the Wnt signaling cascade. In addition, Apc plays an important role in multiple cellular functions, including cell migration and adhesion, spindle assembly, and chromosome segregation. However, its role during adult hematopoiesis is unknown. We show that conditional inactivation of Apc in vivo dramatically increases apoptosis and enhances cell cycle entry of hematopoietic stem cells (HSCs)/ hematopoietic progenitor cells (HPCs), leading to their rapid disappearance and bone marrow failure. The defect in HSCs/HPCs caused by Apc ablation is cell autonomous. In addition, we found that loss of Apc leads to exhaustion of the myeloid progenitor pool (common myeloid progenitor, granulocyte-monocyte progenitor, and megakaryocyte-erythroid progenitor), as well as the lymphoid-primed multipotent progenitor pool. Down-regulation of the genes encoding Cdkn1a, Cdkn1b, and Mcl1 occurs after acute Apc excision in candidate HSC populations. Together, our data demonstrate that Apc is essential for HSC and HPC maintenance and survival.     

Platelet lysate supports the in vitro expansion of human periodontal ligament stem cells for cytotherapeutic use

Human platelet lysate (PL) produced under optimal conditions of standardization and safety has been increasingly suggested as the future ‘gold standard’ supplement to replace fetal bovine serum (FBS) for the ex vivo propagation of mesenchymal stem cells for translational medicine and cell therapy applications. However, the multifaceted effects of PL on tissue‐specific stem cells remain largely unexplored. In the present study, we investigated the stem cell behaviours of human periodontal ligament stem cells (PDLSCs) in media with or without PL. Our data indicate that human PL, either as an adjuvant for culture media or as a substitute for FBS, supports the proliferation and expansion of human PDLSCs derived from either ‘young’ or ‘old’ donors to the same extent as FBS, without interfering with their immunomodulatory capacities. Although PL appears to inhibit the in vitro differentiation of ‘young’ or ‘old’ PDLSCs, their decreased osteogenic potential may be restored to similar or higher levels compared with FBS‐expanded cells. PL‐ and FBS‐expanded PDLSCs exhibited a similar potential to form mineralized nodules and expressed similar levels of osteogenic genes. Our data indicate that large clinically relevant quantities of PDLSCs may be yielded by the use of human PL; however, further analysis of its precise composition and function will pave the way for determining optimized, defined culture conditions. In addition to the potential increase in patient safety, our findings highlight the need for further research to develop the potential of PL‐expanded PDLSCs for clinical use. Copyright © 2016 John Wiley & Sons, Ltd.     

HOXC10 promotes proliferation and attenuates lipid accumulation of sheep bone marrow mesenchymal stem cells

Homeodomain-containing gene C10 (HOXC10), known to regulate cell differentiation and proliferation, is a key negative regulator in the browning of white adipose tissue in mice. Sheep is an important farm animal that provides meat for human consumption, with fat content being an important meat quality determinant; however, there is no report about the role of HOXC10 in sheep adipocytes or adipogenesis. In this study, we investigated the effect of HOXC10 on proliferation and adipogenic differentiation in sheep bone marrow mesenchymal stem cells (sBMSCs). In sBMSCs, HOXC10 overexpression promoted cell proliferation and upregulated the expression of p-PI3K, p-AKT, p-p70S6K, p-MEK, and p-ERK, whereas HOXC10 knockdown was associated with the opposite effects. These results suggested that HOXC10 may promote cell proliferation by activating the MEK/ERK and PI3K/AKT/mTOR/p70S6K signaling pathways. In addition, we found that HOXC10 expression was negatively associated with lipid accumulation in adipogenic-differentiated sBMSCs. HOXC10 overexpression in sBMSCs significantly decreased lipid droplet accumulation and suppressed the expression of adipogenic-specific genes, including ACC, LPL, PPARG, and FABP4, while HOXC10 knockdown was associated with the opposite effects. Furthermore, our study suggested a new regulatory mechanism of the effect of HOXC10 on lipid accumulation and metabolism; HOXC10 may negatively regulate lipid accumulation in adipogenic-differentiated sBMSCs, at least in part, by suppressing LPL expression. Overall, our research not only contributes to a better understanding of the mechanism of lipid accumulation and metabolism in sheep, but also shed light on meat quality control in the future.     

Targeted neural differentiation of murine mesenchymal stem cells by a protocol simulating the inflammatory site of neural injury

Damaged neural tissue is regenerated by neural stem cells (NSCs), which represent a rare and difficult‐to‐culture cell population. Therefore, alternative sources of stem cells are being tested to replace a shortage of NSCs. Here we show that mouse adipose tissue‐derived mesenchymal stem cells (MSCs) can be effectively differentiated into cells expressing neuronal cell markers. The differentiation protocol, simulating the inflammatory site of neural injury, involved brain tissue extract, fibroblast growth factor, epidermal growth factor, supernatant from activated splenocytes and electrical stimulation under physiological conditions. MSCs differentiated using this protocol displayed neuronal cell morphology and expressed genes for neuronal cell markers, such as neurofilament light (Nf‐L), medium (Nf‐M) and heavy (Nf‐H) polypeptides, synaptophysin (SYP), neural cell adhesion molecule (NCAM), glutamic acid decarboxylase (GAD), neuron‐specific nuclear protein (NeuN), βIII‐tubulin (Tubb3) and microtubule‐associated protein 2 (Mtap2), which are absent (Nf‐L, Nf‐H, SYP, GAD, NeuN and Mtap2) or only slightly expressed (NCAM, Tubb3 and Nf‐M) in undifferentiated cells. The differentiation was further enhanced when the cells were cultured on nanofibre scaffolds. The neural differentiation of MSCs, which was detected at the level of gene expression, was confirmed by positive immunostaining for Nf‐L protein. The results thus show that the simulation of conditions in an injured neural tissue and inflammatory environment, supplemented with electrical stimulation under physiological conditions and cultivation of cells on a three‐dimensional (3D) nanofibre scaffold, form an effective protocol for the differentiation of MSCs into cells with neuronal markers. Copyright © 2015 John Wiley & Sons, Ltd.     

Humanized neurofibroma model from induced pluripotent stem cells delineates tumor pathogenesis and developmental origins

Neurofibromatosis type 1 (NF1) is a common tumor predisposition syndrome caused by NF1 gene mutation, in which affected patients develop Schwann cell lineage peripheral nerve sheath tumors (neurofibromas). To investigate human neurofibroma pathogenesis, we differentiated a series of isogenic, patient-specific NF1-mutant human induced pluripotent stem cells (hiPSCs) into Schwannian lineage cells (SLCs). We found that, although WT and heterozygous NF1-mutant hiPSCs-SLCs did not form tumors following mouse sciatic nerve implantation, NF1-null SLCs formed bona fide neurofibromas with high levels of SOX10 expression. To confirm that SOX10⁺ SLCs contained the cells of origin for neurofibromas, both Nf1 alleles were inactivated in mouse Sox10⁺ cells, leading to classic nodular cutaneous and plexiform neurofibroma formation that completely recapitulated their human counterparts. Moreover, we discovered that NF1 loss impaired Schwann cell differentiation by inducing a persistent stem-like state to expand the pool of progenitors required to initiate tumor formation, indicating that, in addition to regulating MAPK-mediated cell growth, NF1 loss also altered Schwann cell differentiation to promote neurofibroma development. Taken together, we established a complementary humanized neurofibroma explant and, to our knowledge, first-in-kind genetically engineered nodular cutaneous neurofibroma mouse models that delineate neurofibroma pathogenesis amenable to future therapeutic target discovery and evaluation.     

The CDK inhibitors: potential targets for therapeutic stem cell manipulations?

Therapies involving adult stem cells are dependent upon sufficient expansion of these cells to repopulate or replace the diseased tissue and are consequently hindered by their relatively quiescent phenotype. Cellular proliferation is governed by the cyclin-dependent kinases, which in a complex with a corresponding cyclin, phosphorylate a number of downstream mediators to drive the cell through the cell cycle. In turn, biochemical activities of the cyclin-dependent kinases are regulated by two families of cyclin-dependent kinase inhibitors, which have been shown to be potent cell intrinsic blocks of adult stem cell proliferation in multiple tissue types. In contrast to normal stem cells, inappropriate regulation of the cell cycle in cancer stem cells may underlie tumorigenesis and failure of conventional chemotherapeutics to fully eradicate a tumor. Thus, definition of the roles of the cyclin-dependent kinase inhibitors in normal and cancer stem cells may permit the development of novel strategies for adult stem cell expansion and therapies specifically targeted to cancer stem cells.     

QUANTITATIVE STUDIES ON THE MIXED LYMPHOCYTE INTERACTION IN RATS : VI. REACTIVITY OF LYMPHOCYTES FROM CONVENTIONAL AND GERMFREE RATS TO ALLOGENEIC AND XENOGENEIC CELL SURFACE ANTIGENS

The proliferative reactivity of lymphocytes from rat donors maintained under germfree or conventional conditions was examined in mixed lymphocyte cultures stimulated with allogeneic and xenogeneic cell surface antigens. The results show (a) that lymphocytes from conventionally maintained rats are less reactive to human, hamster, guinea pig, and mouse cell surface antigens than to the major H alloantigens, and (b) that lymphocytes from germfree rats display no demonstrable reactivity to xenogeneic cells, but are quantitatively normal in their response to allogenic cells. The conclusion drawn from these observations is that the circulating lymphocyte pool of an individual consists of a greater proportion of cells reactive to H alloantigens of other members of the same species than to the xenogeneic cellular antigens of members of other species and that this large number of cells is not generated by a mechanism involving immunization to cross-reactive environmental antigens.