Vascularized Lung Cancer Model for Evaluating the Promoted Transport of Anticancer Drugs and Immune Cells in an Engineered Tumor Microenvironment.

The tumor microenvironment (TME) is the environment around the tumor, including blood vessels, immune cells, fibroblasts, signaling molecules, and the extracellular matrix (ECM). Owing to its component interactions, the TME influences tumor growth and drug delivery in a highly complex manner. Although several vascularized cancer models are developed to mimic the TME in vitro, these models cannot comprehensively reflect blood vessel-tumor spheroid interactions. Here, a method for inducing controlled tumor angiogenesis by engineering the microenvironment is presented. The interstitial flow direction regulates the direction of capillary sprouting, showing that angiogenesis occurs in the opposite direction of flow, while the existence of lung fibroblasts affects the continuity and lumen formation of sprouted capillaries. The vascularized tumor model shows enhanced delivery of anticancer drugs and immune cells to the tumor spheroids because of the perfusable vascular networks. The possibility of capillary embolism using anticancer drug-conjugated liquid metal nanoparticles is investigated using the vascularized tumor model. This vascularized tumor platform can aid in the development of effective anticancer drugs and cancer immunotherapy.

An engineered neurovascular unit for modeling neuroinflammation.

The neurovascular unit (NVU) comprises multiple types of brain cells, including brain endothelial cells, astrocytes, pericytes, neurons, microglia, and oligodendrocytes. Each cell type contributes to the maintenance of the molecular transport barrier and brain tissue homeostasis. Several disorders and diseases of the central nervous system, including neuroinflammation, Alzheimer's disease, stroke, and multiple sclerosis, have been associated with dysfunction of the NVU. As a result, there has been increased demand for the development of NVUin vitromodels. Here, we present a three-dimensional (3D) immortalized human cell-based NVU model generated by organizing the brain microvasculature in a collagen matrix embedded with six different types of cells that comprise the NVU. By surrounding a perfusable brain endothelium with six types of NVU-composing cells, we demonstrated a significant impact of the 3D co-culture on the maturation of barrier function, which is supported by cytokines secreted from NVU-composing cells. Furthermore, NVU-composing cells alleviated the inflammatory responses induced by lipopolysaccharides. Our human cell-based NVUin vitromodel could enable elucidation of both physiological and pathological mechanisms in the human brain and evaluation of safety and efficacy in the context of high-content analysis during the process of drug development.

Microphysiological systems for recapitulating physiology and function of blood-brain barrier.

Central nervous system (CNS) diseases are emerging as a major issue in an aging society. Although extensive research has focused on the development of CNS drugs, the limited transport of therapeutic agents across the blood-brain barrier (BBB) remains a major challenge. Conventional two-dimensional culture dishes do not recapitulate in vivo physiology and real-time observations of molecular transport are not possible in animal models. Recent advances in engineering techniques have enabled the generation of more physiologically relevant in vitro BBB models, and their applications have expanded from fundamental biological research to practical applications in the pharmaceutical industry. In this article, we provide an overview of recent advances in the development of in vitro BBB models, with a particular focus on the recapitulation of BBB function. The development of biomimetic BBB models is postulated to revolutionize not only fundamental biological studies but also drug screening.

Cordycepin-enriched Cordyceps militaris induces immunomodulation and tumor growth delay in mouse-derived breast cancer.

Cordyceps militaris (C. militaris) and its main functional component, cordycepin, has been shown to possess a number of pharmacological activities including immunological stimulation and antitumor effects. However, the pharmacological mechanisms of C. militaris on tumor immunity underlying its antitumor effect have yet to be elucidated. In the present study, we evaluated the antitumor and immunomodulatory effects of C. militaris on FM3A tumor-bearing C3H/He mice, comparing wild-type C. militaris and cordycepin-enriched C. militaris (JLM 0636). The concentration of cordycepin produced by crossbred JLM 0636 was 7.42 mg/g dry weight, which was 7-fold higher than that of wild-type C. militaris. Dietary administration of C. militaris revealed retardation of tumor growth as well as elongation of survival rates of tumor-bearing mice. This effect was more pronounced in JLM 0636. There was a cordycepin-dependent decrease in IL-2 and TGF-ß secretion and an increase in IL-4 secretion without changes in the proliferative responses of concanavalin A-stimulated lymphocytes, which suggested that C. militaris feeding might induce changes in the subpopulations of tumor-derived T lymphocytes. CD4+CD25+ cell population was significantly reduced in the total splenocytes from JLM 0636-administered mice, while CD4+ T cell population remained unchanged. FoxP3+-expressing Treg cells among CD4+CD25+ population showed a similar pattern. On the contrary, CD8+ T cells as well as the IFN-γ expressing CD8+ T cells from tumor-bearing mice were significantly upregulated by the administration of JLM 0636. These results demonstrated the suppressive role of JLM 0636 on the function of Treg cells contributing to tumor specific IFN-γ-expressing CD8+ T cell responses in tumor-bearing mice, which explained the underlying mechanism of the antitumor immunity of cordycepin. Therefore, cordycepin-enriched C. militaris is a promising candidate for an adjuvant in cancer immunotherapy.

Inhibitory effects of Asterina pectinifera extracts on melanin biosynthesis through tyrosinase activity.

The control of melanogenesis is an important strategy in the treatment of abnormal skin pigmentation for cosmetic purposes. The aim of the present study was to investigate the anti-melanogenic effect of Asterina pectinifera (A. pectinifera) extracts by cell-free mushroom tyrosinase assay, cellular tyrosinase assay, melanin content assay and the analysis of related protein expression in melan-a cells. A. pectinifera was extracted with 80% methanol (80-MAP) and further fractionated with hexane (He-AP) and ethyl acetate (EA-AP). In addition, the enzyme extract (En-AP) of A. pectinifera, to which protease was added, was processed. EA-AP and En-AP among A. pectinifera extracts showed strong inhibitory activity against the cell-free mushroom tyrosinase activity. EA-AP and En-AP induced significant inhibition of melanin production and cellular tyrosinase activity. In the action of EA-AP and En-AP on melanogenesis, they reduced the expression of melanogenic genes and proteins including tyrosinase, tyrosinase-related protein-1 (TRP-1) and dopachrome tautomerase (Dct). These results showed that EA-AP and En-AP inhibited melanogenesis by reducing tyrosinase activity and melanin production via subsequent downregulation of tyrosinase-related proteins. The overall results suggest that EA-AP and En-AP among A. pectinifera extracts may be promising candidates for the treatment of hyperpigmentation disorder and useful for self-tanning cosmetic products.

Mesenchymal stem cells overexpressing GCP-2 improve heart function through enhanced angiogenic properties in a myocardial infarction model.

AIMS: In this study, our aim was to evaluate the angio-vasculogenic properties of human adipose tissue-derived mesenchymal stem cells overexpressing the granulocyte chemotactic protein (GCP)-2 (hASCs/GCP-2) and to determine possible therapeutic effects in an experimental ischaemic heart model. METHODS AND RESULTS: Quantitative real-time (qRT)-PCR results revealed that hASCs/GCP-2 expressed significantly higher levels of pro-angiogenic genes, including vascular endothelial growth factor (VEGF)-A, hepatocyte growth factor (HGF), and interleukin (IL)-8, when compared with control-vector transduced hASCs or human umbilical vascular endothelial cells (HUVECs). In addition, the anti-apoptotic insulin-like growth factor (IGF)-1 and Akt-1 were also highly up-regulated in the hASCs/GCP-2 cells. In vitro cell migration and proliferation assays showed that hASCs/GCP-2-derived conditioned media (CM) significantly accelerated the migration and proliferation of fibroblast cells. Examination of in vitro endothelial differentiation showed that hASCs/GCP-2 cells spontaneously formed vascular-like structures and highly expressed endothelial-specific genes and proteins. In vivo study results of our mouse myocardial infarction (MI) model revealed that hASCs/GCP-2 implantation improved the cardiac function and reduced the infarct size. Finally, transplanted hASCs/GCP-2 cells unexpectedly differentiated into endothelial cells and the engraftment rate was significantly higher than control groups. CONCLUSION: We suggest that overexpression of GCP-2 in stem cells has the potential to enhance their angiogenic and survival properties.

Pathological adaptive responses of Schwann cells to endoplasmic reticulum stress in bortezomib-induced peripheral neuropathy.

Bortezomib, a proteasome inhibitor, has been considered as a promising anticancer drug in the treatment of recurrent multiple myeloma and some solid tumors. The bortezomib-induced peripheral neuropathy (BIPN) is a prominent cause of dose-limiting toxicities after bortezomib treatment. In this study, we found that BIPN in a mouse model is characterized by acute but transient endoplasmic reticulum (ER) damages to Schwann cells. These damaged Schwann cells exhibit abnormal outcomes from healing processes such as the myelination of Remak bundles. A morphometric analysis of polymyelinated Remak bundles revealed that the pathological myelination was not related to the axonal parameters that regulate the normal myelination process during development. In addition, demyelinating macrophages were focally infiltrated within endoneurium of the sciatic nerve. To identify the mechanism underlying these pathologies, we applied a gene microarray analysis to bortezomib-treated primary Schwann cells and verified the changes of several gene expression in bortezomib-treated sciatic nerves. The analysis showed that bortezomib-induced ER stress was accompanied by the activation of several protective molecular chaperones and the down-regulation of myelin gene expression. ER stress inducers such as thapsigargin and bredelfin A also suppressed the mRNA expression of myelin gene P0 at transcriptional levels. In addition, the expression of chemokines such as the macrophage chemoattractants Ccl3 and Cxcl2 was significantly increased in Schwann cells in response to bortezomib and ER stress inducers. Taken together, these observations suggest that the pathological adaptive responses of Schwann cells to bortezomib-induced ER stress may, in part, participate in the development of BIPN.

Injury-induced CRMP4 expression in adult sensory neurons; a possible target gene for ciliary neurotrophic factor.

Neurotrophic cytokines, such as ciliary neurotrophic factor (CNTF) play an important role in the development and regeneration of the nervous system. In the present study, we screened gene expression induced by CNTF in adult dorsal root ganglion (DRG) neurons using the Illumina microarray. We found that the expression of both short and long forms of collapsin response-mediator protein 4 (CRMP4) was increased in cultured primary sensory neurons by CNTF. In addition, sciatic nerve injury induced the expression of CRMP4 mRNA and protein in DRG neurons. Finally, the increased CRMP4 protein was transported into peripheral axons following nerve injury. These findings indicate that CRMP4 may be a target gene for CNTF in the regenerative axon growth of DRG neurons after injury.

Fenofibrate Reduces Age-related Hypercholesterolemia in Normal Rats on a Standard Diet.

Plasma cholesterol is increased in normal aging in both rodents and humans. This is associated with reduced elimination of cholesterol and decreased receptor-mediated clearance of plasma low-density lipoprotein (LDL) cholesterol. The aims of this study were: (1) to determine age-related changes in plasma lipid profiles, and (2) to determine the effect of fenofibrate, an activator of peroxisome proliferator activated receptor alpha (PPAR alpha), on plasma lipid profiles in normal rats on a standard diet. Male Sprague-Dawley (SD) rats (n=15) were fed standard chow and water from 10 to 25 weeks of age. During that period, we measured daily food intake, body weight, fasting and random blood glucose levels, plasma total cholesterol (TC), triglycerides (TG), and free fatty acid (FFA) levels. At 20 weeks of age, all rats were randomly divided into two groups: a fenofibrate group (in which rats were gavaged with 300 mg/kg/day of fenofibrate) and a control group (gavaged with water). Fenofibrate treatment lasted 5 weeks. There were no significant changes in daily food intake, blood glucose, and plasma TG level with age. Body weight, plasma TC, and FFA levels were significantly increased with age. Fenofibrate significantly decreased plasma concentrations of TC and FFA, which had been increased with age. However, fenofibrate did not influence the plasma concentration of TG, which had not increased with age. These results suggest that fenofibrate might have a novel role in preventing age-related hypercholesterolemia in SD rats on a normal diet.

Janus Kinase 2 Inhibitor AG490 Inhibits the STAT3 Signaling Pathway by Suppressing Protein Translation of gp130.

The binding of interleukin-6 (IL-6) cytokine family ligands to the gp130 receptor complex activates the Janus kinase (JAK)/ signal transducer and activator of transcription 3 (STAT3) signal transduction pathway, where STAT3 plays an important role in cell survival and tumorigenesis. Constitutive activation of STAT3 has been frequently observed in many cancer tissues, and thus, blocking of the gp130 signaling pathway, at the JAK level, might be a useful therapeutic approach for the suppression of STAT3 activity, as anticancer therapy. AG490 is a tyrphostin tyrosine kinase inhibitor that has been extensively used for inhibiting JAK2 in vitro and in vivo. In this study, we demonstrate a novel mechanism associated with AG490 that inhibits the JAK/STAT3 pathway. AG490 induced downregulation of gp130, a common receptor for the IL-6 cytokine family compounds, but not JAK2 or STAT3, within three hours of exposure. The downregulation of gp130 was not caused by enhanced degradation of gp130 or by inhibition of mRNA transcription. It most likely occurred by translation inhibition of gp130 in association with phosphorylation of the eukaryotic initiation factor-2alpha . The inhibition of protein synthesis of gp130 by AG490 led to immediate loss of mature gp130 in cell membranes, due to its short half-life, thereby resulting in reduction in the STAT3 response to IL-6. Taken together, these results suggest that AG490 blocks the STAT3 activation pathway via a novel pathway.

Extracellular Signal-regulated Kinase Activation Is Required for Serine 727 Phosphorylation of STAT3 in Schwann Cells in vitro and in vivo.

In the peripheral nerves, injury-induced cytokines and growth factors perform critical functions in the activation of both the MEK/ERK and JAK/STAT3 pathways. In this study, we determined that nerve injury-induced ERK activation was temporally correlated with STAT3 phosphorylation at the serine 727 residue. In cultured Schwann cells, we noted that ERK activation is required for the serine phosphorylation of STAT3 by neuropoietic cytokine interleukin-6 (IL-6). Serine phosphorylated STAT3 by IL-6 was transported into Schwann cell nuclei, thereby indicating that ERK may regulate the transcriptional activity of STAT3 via the induction of serine phosphorylation of STAT3. Neuregulin-1 (NRG) also induced the serine phosphorylation of STAT3 in an ERK-dependent fashion. In contrast with the IL-6 response, serine phosphorylated STAT3 induced by NRG was not detected in the nucleus, thus indicating the non-nuclear function of serine phosphorylated STAT3 in response to NRG. Finally, we determined that the inhibition of ERK prevented injury-induced serine phosphorylation of STAT3 in an ex-vivo explants culture of the sciatic nerves. Collectively, the results of this study show that ERK may be an upstream kinase for the serine phosphorylation of STAT3 induced by multiple stimuli in Schwann cells after peripheral nerve injury.

Proteasome inhibition suppresses Schwann cell dedifferentiation in vitro and in vivo.

The ubiquitin-proteasome system (UPS), lysosomes, and autophagy are essential protein degradation systems for the regulation of a variety of cellular physiological events including the cellular response to injury. It has recently been reported that the UPS and autophagy mediate the axonal degeneration caused by traumatic insults and the retrieval of nerve growth factors. In the peripheral nerves, axonal degeneration after injury is accompanied by myelin degradation, which is tightly related to the reactive changes of Schwann cells called dedifferentiation. In this study, we examined the role of the UPS, lysosomal proteases, and autophagy in the early phase of Wallerian degeneration of injured peripheral nerves. We found that nerve injury induced an increase in the ubiquitin conjugation and lysosomal-associated membrane protein-1 expression within 1 day without any biochemical evidence for autophagy activation. Using an ex vivo explant culture of the sciatic nerve, we observed that inhibiting proteasomes or lysosomal serine proteases prevented myelin degradation, whereas this was not observed when inhibiting autophagy. Interestingly, proteasome inhibition, but not leupeptin, prevented Schwann cells from inducing dedifferentiation markers such as p75 nerve growth factor receptor and glial fibrillary acidic protein in vitro and in vivo. In addition, proteasome inhibitors induced cell cycle arrest and cellular process formation in cultured Schwann cells. Taken together, these findings indicate that the UPS plays a role in the phenotype changes of Schwann cells in response to nerve injury.

Netrin-1 induces proliferation of Schwann cells through Unc5b receptor.

Netrin and its receptors, DCC (Deleted in Colorectal Cancer) and Unc5, are proposed to be involved in the axon guidance and neuroglial migration during development. However, accumulating evidence implies that they may also participate in the cell survival and apoptosis. Here, we show that netrin-1 induces proliferation of Schwann cells. Unc5b is the sole receptor expressed in RT4 schwannoma cells and adult primary Schwann cells, and netrin-1 and Unc5b are found to be expressed in the injured sciatic nerve. It was also found that the netrin-1-induced Schwann cell proliferation was blocked by the specific inhibition of Unc5b expression with RNAi. These data suggest that netrin-1 could be an endogenous trophic factor for Schwann cells in the injured peripheral nerves.

Antitumor activity of TRAIL recombinant adenovirus in human malignant glioma cells.

Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) has been reported to specifically kill malignant cells but to be relatively nontoxic to normal cells. One of disadvantages to previous in vivo protocols was the need for large quantities of TRAIL recombinant protein to suppress tumor growth. To evaluate the antitumor activity and therapeutic value of the TRAIL gene, we constructed adenoviral vectors expressing the human TRAIL gene (Ad.hTRAIL) and transferred them into malignant glioma cells in vitro and tumors in vivo, as an alternative to recombinant soluble TRAIL protein. The results show that TRAIL-sensitive glioma cells infected Ad.hTRAIL undergo apoptosis through the production and expression of TRAIL protein. The in vitro transfer elicited apoptosis, as demonstrated by the quantification of viable or apoptotic cells and by the analysis of cleavage of poly (ADP-ribose) polymerase. Furthermore, in vivo administration of Ad.hTRAIL at the site of tumor implantation suppressed the outgrowth of human glioma xenografts in SCID mice. These results further define Ad.hTRAIL as an anti-tumor therapeutic and demonstrate its potential use as an alternative approach to treatment for malignant glioma.

Stem cells expressing homing receptors could be expanded from cryopreserved and unselected cord blood.

We assessed the cytokine combinations that are best for ex vivo expansion of cord blood (CB) and the increment for cell numbers of nucleated cells, as well as stem cells expressing homing receptors, by an ex vivo expansion of cryopreserved and unselected CB. Frozen leukocyte concentrates (LC) from CB were thawed and cultured at a concentration of 1 x 10(5)/mL in media supplemented with a combination of SCF (20 ng/mL)+TPO (50 ng/mL)+FL (50 ng/mL)+/-IL-6 (20 ng/mL)+/-G-CSF (20 ng/mL). After culturing for 14 days, the expansion folds of cell numbers were as follows: TNC 22.3+/-7.8 approximately 26.3+/-4.9, CFU-GM 4.7+/-5.1 approximately 11.7+/-2.6, CD34+CD38- cell 214.0+/-251.9 approximately 464.1+/-566.1, CD34+CXCR4+ cell 4384.5+/-1664.7 approximately 7087.2+/-4669.3, CD34+VLA4+ cell 1444.3+/-1264.0 approximately 2074.9+/-1537.0, CD34+VLA5+ cell 86.2+/-50.9 approximately 113.2+/-57.1. These results revealed that the number of stem cells expressing homing receptors could be increased by an ex vivo expansion of cryopreserved and unselected CB using 3 cytokines (SCF, TPO, FL) only. Further in vivo studies regarding the engraftment after expansion of the nucleated cells, as well as the stem cells expressing homing receptors will be required.

Establishment and characterization of an STI571-resistant human myelogenous leukemia cell line, SR-1.

The tyrosine kinase inhibitor STI571 is an effective agent for the treatment of chronic myelogenous leukemia (CML). However, a lack of response to STI571 or the recurrence of the disease after a transient initial response is usually seen in patients with advanced stage CML. We have established a novel STI571 (Gleevec/Glivec, imatinib mesylate)-resistant acute myelocytic leukemia cell line (SR-1) from an STI571-resistant blast crisis patient. By flow cytometry, the immunophenotype of SR-1 was found to be compatible with a myeloid lineage (CD13+, CD33+, HLA-DR+, anti-MPO+). Conventional cytogenetics showed a three-way reciprocal translocation involving 7p22, 9q34, and 22q11.2, i.e., a variant Philadelphia chromosome translocation. The BCR/ABL rearrangement was detected by fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction. To determine the tumorigenicity of the SR-1 cell line in vivo, cells were injected subcutaneously into severe combined immunodeficiency mice. Four weeks later, tumors had grown and showed the same laboratory findings as in SR-1. Although STI571 resistance is a known treatment complication, in vivo STI571-resistant cell lines have not been fully established. We hope that our SR-1 cell line may be useful in molecular pathogenetic investigations of STI571-resistant CML.

Homing-associated cell adhesion molecules and cell cycle status on the nucleated cells in the bone marrow, mobilized peripheral blood and cord blood.

Homing-associated cell adhesion molecules (H-CAM) on the CD34+ cells play an important role for the engraftment process following hematopoietic stem cell transplantation (HSCT). However, it seems that not only CD34+ cells but also other nucleated cells (NCs) with H-CAM could be implicated in the engraftment process and the proliferation of hematopoietic stem cells. We investigated the differences of HCAM and cell cycle status on the NCs in cord blood (CB), bone marrow (BM), and mobilized peripheral blood (PB). The proportions of CXCR4+ cells within the NC populations were greater in CB than in PB or BM (p=0.0493), although the proportions of CXCR4+, CD44+, and CD49d+ cells within the CB CD34+ cell populations were same within BM or PB. A lower proportion of CD34+CD49d+ cells within the CD34+ cell populations was more noted in CB than in PB or BM (p=0.0085). There were no differences in cell cycle status between CB and BM or PB. Our results suggest that the migrating potential of CB would be enhanced with increased CXCR4 expression on the NCs, but the adhesion potential of CB CD34+ cells would be less than that of PB and BM. These findings may help explain why the lower cell dose is required and engraftment is delayed in cord blood stem cell transplantation.

Dendritic cells loaded with exogenous antigen by electroporation can enhance MHC class I-mediated antitumor immunity.

To develop an efficient antitumor immunotherapy, we have examined if dendritic cells (DCs) loaded with soluble antigens by electroporation present more antigens via the MHC (major histocompatibility complex) class I pathway, which mediate a cytotoxic T-cell response. DCs loaded with ovalbumin (OVA) by electroporation presented more MHC class I-restricted determinants compared with DCs pulsed with OVA. When electroporated DCs were pulsed with OVA for additional times, both MHC class I- and II-restricted presentation of OVA were increased compared with each single procedure, including electroporation or simple pulse. Immunization with DCs loaded with OVA by electroporation induced higher cytotoxicity of splenocytes to E.G7 cells, a clone of EL4 cells transfected with an OVA cDNA, than immunization with DCs pulsed with OVA. In the animal study, immunization with DCs loaded with OVA or tumor cell lysates by electroporation induced an effective antitumor immunity against tumor of E.G7 cells or Lewis lung carcinoma cells, respectively. In addition, immunization with DCs loaded with antigen by combination of electroporation and pulse, completely protected mice from tumor formation, and prolonged survival, in both tumor models. These results demonstrated that electroporation would be a useful way to enhance MHC class I-mediated antitumor immunity without functional deterioration, and that the combination of electroporation and pulse could be a simple and efficient antigen-loading method and consequently lead to induction of strong antitumor immunity.

A single administration of adenoviral-mediated HGF cDNA permits survival of mice from acute hepatic failure.

Heptatocyte growth factor (HGF) having a variety of biological activity was suggested as a protective agent against acute toxic hepatic injury or a potentially therapeutic agent. For the efficient in vivo application of this factor, we employed adenoviral-mediated HGF gene delivery system. In this study, we constructed E1-deleted recombinant adenovirus carrying cDNA of human HGF (Ad.hHGF) and elucidated that HGF was efficiently expressed in the liver of C57/BL mice. A mouse model of acute hepatic failure was induced by high dose (1000mg/kg) of thioacetamide (TA) administration. Mice infected with Ad.hHGF showed a dramatic resistance to TA-induced acute hepatic injury. Serum ALT was increased transiently and then the level was normalized in Ad.hHGF-infected mice with TA administration. Furthermore, the survival rate was remarkably enhanced in the mice infected with Ad.hHGF. In the histological examination, massive hepatic necrosis induced by TA was almost completely protected by HGF produced by Ad.hHGF. Our results indicate that a single dose of HGF-encoding adenoviral vector maintained liver function and prevented the progression of liver necrosis in a mouse model of acute hepatic failure.

Urokinase could increase the yield of progenitor cells during red cell depletion in elapsed and anticoagulated cord blood.

We assessed whether the urokinase could increase the yield of progenitor cells during processing in elapsed, anticoagulated cord blood (CB) after collection, and we also determined the optimal dose of urokinase. The total nucleated cell (TNC) counts after red cell depletion in 48-hr-elapsed CB were significantly higher in samples treated with 10,000 and 50,000 IU of urokinase/mL than in untreated samples or treated with 5,000 IU of urokinase/mL. The CD34(+) cell counts were significantly higher in samples treated with 10,000 IU of urokinase/mL than in untreated samples and in samples treated with 5,000 or 50,000 IU of urokinase/mL. In 6-, 12-, and 24-hr-elapsed CB, however, there were no significant differences of TNC, CD34(+) cells, or CFU-GM counts between untreated samples and samples treated with 10,000 IU of urokinase/mL. These findings suggest that the addition of 10,000 IU of urokinase/mL before red cell depletion in 48-hr-elapsed, anticoagulated CB could increase the yield of progenitor cells. However, there are no advantages in using urokinase for processing CB prior to 24 hr after collection.