Differentiated Cells Derived from Hematopoietic Stem Cells and Their Applications in Translational Medicine.

Hematopoietic stem cells (HSCs) and their development are one of the most widely studied model systems in mammals. In adults, HSCs are predominantly found in the bone marrow, from where they maintain homeostasis. Besides bone marrow and mobilized peripheral blood, cord blood is also being used as an alternate allogenic source of transplantable HSCs. HSCs from both autologous and allogenic sources are being applied for the treatment of various conditions like blood cancers, anemia, etc. HSCs can further differentiate to mature blood cells. Differentiation process of HSCs is being extensively studied so as to obtain a large number of pure populations of various differentiated cells in vitro so that they can be taken up for clinical trials. The ability to generate sufficient quantity of clinical-grade specialized blood cells in vitro would take the field of hematology a step ahead in translational medicine.

Flow Cytometry and Cell Sorting Using Hematopoietic Progenitor Cells.

Flow cytometry is a widely used laser-based technology for rapid analysis of the expression of cell surface antigens and intracellular molecules in various cell types including hematopoietic stem/progenitor cells (HSPCs). Multiparametric analysis of individual cells within a short time frame makes this tool attractive and indispensable in the field of stem cell research. This is accomplished by harnessing the specific light scattering ability of the cell type, which determines its size and internal complexity. In addition, use of fluorescently conjugated antibodies allows the detection of a specific surface or intracellular antigen present at that particular stage. Fluorescent Activated Cell Sorting (FACS) is used to separate a subset of cells from a heterogeneous cell population based on fluorescent labeling. Here we describe the general principles of flow cytometry and detailed methods for the isolation of HSPCs using flow cytometry as a tool.

Neuropilin-1 Is an Important Niche Component and Exerts Context-Dependent Effects on Hematopoietic Stem Cells.

Marrow adipocytes pose a significant problem in post-transplant regeneration of hematopoiesis owing to their negative effects on regeneration of hematopoiesis. However, the precise mechanism operative in this negative regulation is not clear. In this study, we show that marrow adipocytes express neuropilin-1 (NRP1) as a function of differentiation and inhibit regeneration of hematopoiesis by three principal mechanisms: one, by inducing apoptosis in hematopoietic stem/progenitor cells (HSPCs) through the death receptor-mediated pathway; two, by downregulating CXCR4 expression on the HSPCs through ligand-mediated internalization; and three, by secreting copious amounts of transforming growth factor ß1 (TGFß1), a known inhibitor of hematopoiesis. Silencing of NRP1 in these adipocytes rescued the apoptosis of cocultured HSPCs and boosted the CXCR4 surface expression on them, showing an active role of NRP1 in these processes. However, such silencing had no effect on TGFß1 secretion and consequent inhibition of hematopoiesis by them, showing that secretion of TGFß1 by adipocytes is independent of NRP1 expression by them. Surprisingly, mesenchymal stromal cells modified with NRP1 supported expansion of HSPCs having enhanced functionality, suggesting that NRP1 exerts a context-dependent effect on hematopoiesis. Our data demonstrate that NRP1 is an important niche component and exerts context-dependent effects on HSPCs. Based on these data, we speculate that antibody- or peptide-mediated blocking of NRP1-HSC interactions coupled with a pharmacological inhibition of TGFß1 signaling may help in combating the negative regulation of post-transplant regeneration of hematopoiesis in a more effective manner.

Irradiation-induced secretion of BMP4 by marrow cells causes marrow adipogenesis post-myelosuppression.

Pre-transplant myeloablation is associated with marrow adipogenesis, resulting in delayed engraftment of hematopoietic stem cells (HSCs). This is strongly undesirable, especially when the donor HSCs are fewer in numbers or have compromised functionality. The molecular mechanisms behind irradiation-induced marrow adipogenesis have not been extensively investigated. Here we show that bone marrow (BM) cells, especially T-cells and stromal cells, express and secrete copious amounts of BMP4 in response to irradiation, which causes the bone marrow stromal cells to commit to adipocyte lineage, thereby contributing to an increase in bone marrow adipogenesis. We further demonstrate that Simvastatin inhibits the BMP4-mediated adipogenic commitment of marrow stromal cells by inhibiting Ppar-γ expression. Importantly, Simvastatin does not prevent BMP4 secretion by the BM cells, and thus does not interfere with its salutary role in post-transplant hematopoietic regeneration. Our data identify previously unknown mechanisms operative in marrow adipogenesis post-myeloablation. They also reveal the molecular mechanisms behind the advantage of using Simvastatin as a niche-targeting agent to improve HSC engraftment.

Placenta-derived mesenchymal stem cells possess better immunoregulatory properties compared to their cord-derived counterparts-a paired sample study.

Mesenchymal stem cells (MSCs) show immunoregulatory properties. Here, we compared MSCs obtained from placenta (P-MSCs) and umbilical cord (C-MSCs) from the same donor, for their immunomodulatory efficacy. P-MSCs and C-MSCs showed similar morphology and phenotypic profile, but different clonogenic ability. Importantly, they showed a significant difference in their immunosuppressive properties as assessed in mixed leukocyte reaction (MLR). The P-MSCs affected the antigen presenting ability of mononuclear cells (MNCs) and dendritic cells (DCs) significantly as compared to C-MSCs resulting in a reduced T-cell proliferation. P-MSC conditioned medium (CM) showed a significant reduction in T cell proliferation as compared to C-MSC CM, thus suggesting that a cell to cell contact is not essential. We found increased levels of IL-10 and TGFß1 and reduction in levels of IFNγ in P-MSC MLRs as compared to C-MSC MLRs. Furthermore, the CD3(+) CD4(+) CD25(+) T regulatory cells were enriched in case of P-MSCs in both, MSC-MNC and MSC-DC co-cultures. This observation was further supported by increased mRNA expression of FoxP3 in P-MSCs. Presently, cord-derived MSCs are being employed in transplantation therapies parallel to the bone marrow-derived MSCs. Our findings suggest that P-MSCs can be a better alternative to C-MSCs, to provide aid in immunological ailments.

Pharmacological inhibition of caspase and calpain proteases: a novel strategy to enhance the homing responses of cord blood HSPCs during expansion.

BACKGROUND: Expansion of hematopoietic stem/progenitor cells (HSPCs) is a well-known strategy employed to facilitate the transplantation outcome. We have previously shown that the prevention of apoptosis by the inhibition of cysteine proteases, caspase and calpain played an important role in the expansion and engraftment of cord blood (CB) derived HSPCs. We hypothesize that these protease inhibitors might have maneuvered the adhesive and migratory properties of the cells rendering them to be retained in the bone marrow for sustained engraftment. The current study was aimed to investigate the mechanism of the homing responses of CB cells during expansion. METHODOLOGY/PRINCIPAL FINDINGS: CB derived CD34(+) cells were expanded using a combination of growth factors with and without Caspase inhibitor -zVADfmk or Calpain 1 inhibitor- zLLYfmk. The cells were analyzed for the expression of homing-related molecules. In vitro adhesive/migratory interactions and actin polymerization dynamics of HSPCs were assessed. In vivo homing assays were carried out in NOD/SCID mice to corroborate these observations. We observed that the presence of zVADfmk or zLLYfmk (inhibitors) caused the functional up regulation of CXCR4, integrins, and adhesion molecules, reflecting in a higher migration and adhesive interactions in vitro. The enhanced actin polymerization and the RhoGTPase protein expression complemented these observations. Furthermore, in vivo experiments showed a significantly enhanced homing to the bone marrow of NOD/SCID mice. CONCLUSION/SIGNIFICANCE: Our present study reveals another novel aspect of the regulation of caspase and calpain proteases in the biology of HSPCs. The priming of the homing responses of the inhibitor-cultured HSPCs compared to the cytokine-graft suggests that the modulation of these proteases may help in overcoming the major homing defects prevalent in the expansion cultures thereby facilitating the manipulation of cells for transplant procedures.

Polyunsaturated fatty acids confer cryoresistance on megakaryocytes generated from cord blood and also enhance megakaryocyte production from cryopreserved cord blood cells.

BACKGROUND AIMS: Previous data have shown that the addition of docosahexanoic acid (DHA)/arachidonic acid (AA) has a beneficial effect on cytokine-mediated in vitro generation of megakaryocytes (MK) from umbilical cord blood (UCB).Cryopreservation forms an inherent part of UCB banking and MK progenitors are known to be very sensitive to the stresses of freezing. It is therefore imperative to generate functional cells from cryopreserved cells, and the generated cells need to be cryopreserved until used. In the present study, cryopreservation of ex vivo-expanded MK as well as MK generation from cryopreserved UCB samples was investigated. METHODS: MK generated with or without DHA/AA were cryopreserved in freezing medium containing 10% dimethyl sulfoxide (DMSO). Freezing efficacy was tested by quantitating MK after revival. Cryopreserved CD34(+) cells were cultured with stem cell factor (SCF) and thrombopoietin (TPO), in the presence and absence of DHA/AA for 10 days, and then quantitated for MK. Results. We observed a 1.5-3-fold increase in MK numbers, their progenitor content and their expression of phenotypic markers and MK-related transcription factors. DHA/AA sets showed a 2-5-fold improved engraftment in NOD/SCID mice. These data showed that the beneficial effect of DHA/AA obtained during MK expansion was not altered after freezing stress. The enhancement in MK generation obtained from fresh cord blood (CB) cells was reproduced with comparable efficiency when we used cryopreserved CB samples. CONCLUSIONS: Taken together, our data suggest that in vitro-generated DHA/AA MK survive cryoinjuries in a functionally better state. DHA/AA support a more efficient generation of MK from cryopreserved UCB.

Mimicking the functional hematopoietic stem cell niche in vitro: recapitulation of marrow physiology by hydrogel-based three-dimensional cultures of mesenchymal stromal cells.

BACKGROUND: A culture system that closely recapitulates marrow physiology is essential to study the niche-mediated regulation of hematopoietic stem cell fate at a molecular level. We investigated the key features that play a crucial role in the formation of a functional niche in vitro. DESIGN AND METHODS: Hydrogel-based cultures of human placenta-derived mesenchymal stromal cells were established to recapitulate the fibrous three-dimensional architecture of the marrow. Plastic-adherent mesenchymal stromal cells were used as controls. Human bone marrow-derived CD34(+) cells were co-cultured with them. The output hematopoietic cells were characterized by various stem cell-specific phenotypic and functional parameters. RESULTS: The hydrogel-cultures harbored a large pool of primitive hematopoietic stem cells with superior phenotypic and functional attributes. Most importantly, like the situation in vivo, a significant fraction of these cells remained quiescent in the face of a robust multi-lineage hematopoiesis. The retention of a high percentage of primitive stem cells by the hydrogel-cultures was attributed to the presence of CXCR4-SDF1α axis and integrin beta1-mediated adhesive interactions. The hydrogel-grown mesenchymal stromal cells expressed high levels of several molecules that are known to support the maintenance of hematopoietic stem cells. Yet another physiologically relevant property exhibited by the hydrogel cultures was the formation of hypoxia-gradient. Destruction of hypoxia-gradient by incubating these cultures in a hypoxia chamber destroyed their specialized niche properties. CONCLUSIONS: Our data show that hydrogel-based cultures of mesenchymal stromal cells form a functional in vitro niche by mimicking key features of marrow physiology.

Mannose-binding dietary lectins induce adipogenic differentiation of the marrow-derived mesenchymal cells via an active insulin-like signaling mechanism.

We have recently demonstrated that the mannose-binding lectins, namely banana lectin (BL) and garlic lectin (GL), interacted with the insulin receptors on M210B4 cells--an established mesenchymal cell line of murine marrow origin--and initiate mitogen-activated protein kinase kinase (MEK)-dependent extracellular signal-regulated kinase (ERK) signaling in them. In this study, we show that this lectin-mediated active ERK signaling culminates into an adipogenic differentiation of these cells. Gene expression studies indicate that the effect takes place at the transcriptional level. Experiments carried out with pharmacological inhibitors show that MEK-dependent ERK and phosphatidylinositol 3-kinase-dependent AKT pathways are positive regulators of the lectin- and insulin-mediated adipogenic differentiation, while stress-activated kinase/c-jun N-terminal kinase pathway acts as a negative one. Since both lectins could efficiently substitute for insulin in the standard adipogenic induction medium, they may perhaps serve as molecular tools to study the mechanistic aspects of the adipogenic process that are independent of cell proliferation. Our study clearly demonstrates the ability of BL and GL to activate insulin-like signaling in the mesenchymal cells in vitro leading to their adipocytic differentiation. The dietary origin of these lectins underscores an urgent need to examine their in vivo effects on tissue homeostasis.

Enhanced generation of megakaryocytes from umbilical cord blood-derived CD34(+) cells expanded in the presence of two nutraceuticals, docosahexanoic acid and arachidonic acid, as supplements to the cytokine-containing medium.

BACKGROUND AIMS: Ex vivo generation of megakaryocytes (MK) from hematopoietic stem cells (HSC) is important for both basic research, to understand the mechanism of platelet biogenesis, and clinical infusions, for rapid platelet recovery in thrombocytopenic patients. We investigated the role of two nutraceuticals, docosahexanoic acid (DHA) and arachidonic acid (AA), in the in vitro generation of MK. METHODS: Umbilical cord blood (UCB)-derived CD34+cells were cultured with stem cell factor (SCF) and thrombopoietin (TPO) in the presence (test) or absence (control) of the two additives. On day 10, MK and platelets generated were quantitated by morphologic, phenotypic and functional assays. RESULTS: The cell yield of MK and platelet numbers were significantly higher in test compared with control cells. Phenotypic analyzes and gene expression profiles confirmed these findings. Functional properties, such as colony-forming unit (CFU)-MK formation, chemotaxis and platelet activation, were found to be enhanced in cells cultured with nutraceuticals. The engraftment potential of ex vivo-expanded cells was studied in NOD/SCID mice. Mice that received MK cultured in the presence of DHA/AA engrafted better. There was a reduction in apoptosis and total reactive oxygen species (ROS) levels in the CD41(+) compartment of the test compared with control sets. The data suggest that these compounds probably exert their beneficial effect by modulating apoptotic and redox pathways. CONCLUSIONS: Use of nutraceuticals like DHA and AA may prove to be a useful strategy for efficient generation of MK and platelets from cord blood cells, for future use in clinics and basic research.

Expansion of cord blood CD34 cells in presence of zVADfmk and zLLYfmk improved their in vitro functionality and in vivo engraftment in NOD/SCID mouse.

BACKGROUND: Cord blood (CB) is a promising source for hematopoietic stem cell transplantations. The limitation of cell dose associated with this source has prompted the ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). However, the expansion procedure is known to exhaust the stem cell pool causing cellular defects that promote apoptosis and disrupt homing to the bone marrow. The role of apoptotic machinery in the regulation of stem cell compartment has been speculated in mouse hematopoietic and embryonic systems. We have consistently observed an increase in apoptosis in the cord blood derived CD34(+) cells cultured with cytokines compared to their freshly isolated counterpart. The present study was undertaken to assess whether pharmacological inhibition of apoptosis could improve the outcome of expansion. METHODOLOGY/PRINCIPAL FINDINGS: CB CD34(+) cells were expanded with cytokines in the presence or absence of cell permeable inhibitors of caspases and calpains; zVADfmk and zLLYfmk respectively. A novel role of apoptotic protease inhibitors was observed in increasing the CD34(+) cell content of the graft during ex vivo expansion. This was further reflected in improved in vitro functional aspects of the HSPCs; a higher clonogenicity and long term culture initiating potential. These cells sustained superior long term engraftment and an efficient regeneration of major lympho-myeloid lineages in the bone marrow of NOD/SCID mouse compared to the cells expanded with growth factors alone. CONCLUSION/SIGNIFICANCE: Our data show that, use of either zVADfmk or zLLYfmk in the culture medium improves expansion of CD34(+) cells. The strategy protects stem cell pool and committed progenitors, and improves their in vitro functionality and in vivo engraftment. This observation may complement the existing protocols used in the manipulation of hematopoietic cells for therapeutic purposes. These findings may have an impact in the CB transplant procedures involving a combined infusion of unmanipulated and expanded grafts.

A large number of mature and functional dendritic cells can be efficiently generated from umbilical cord blood-derived mononuclear cells by a simple two-step culture method.

BACKGROUND: Advances in the past two decades in dendritic cell (DC) biology paved the way to exploit them as a promising tool in cancer immunotherapy. The prerequisite for DC vaccine preparations is large-scale in vitro generations of homogeneous, mature, and functional DCs. Frequent improvements are being made in the existing in vitro DC production protocols to achieve this goal. In our previous study we reported a large-scale generation of mature, functional DCs from umbilical cord blood (UCB) CD34+ cells. Here we report that this method can be used for the efficient generation of DCs from UCB mononuclear cells (MNCs) and thus the hematopoietic stem cell isolation step is not essential. STUDY DESIGN AND METHODS: MNCs or CD34+ cells isolated from the same cord blood (CB) samples were used for the generation of DCs. DCs were characterized for morphology, phenotype, and functional assays including antigen uptake, chemotaxis, and mixed leukocyte reaction. Similarly DCs generated from the MNCs of same fresh and frozen CB units were compared. RESULTS: The morphologic, phenotypic, and functional characterization of the DCs generated from various sets show that they were comparable in nature irrespective of the starting population used. CONCLUSION: We conclude that the CD34+ isolation step is not essential for the generation of mature, functional DCs and thus can be eliminated. More importantly, we show that DCs can be generated with equal efficiency from the MNCs of frozen CB units. Our culture method will be useful for exploiting the potential of UCB as an additional source for allogeneic DCs in the clinical settings.

In vitro protection of umbilical cord blood-derived primitive hematopoietic stem progenitor cell pool by mannose-specific lectins via antioxidant mechanisms.

BACKGROUND: Earlier we reported that an oral administration of two mannose-specific dietary lectins, banana lectin (BL) and garlic lectin (GL), led to an enhancement of hematopoietic stem and progenitor cell (HSPC) pool in mice. STUDY DESIGN AND METHODS: Cord blood-derived CD34+ HSPCs were incubated with BL, GL, Dolichos lectin (DL), or artocarpin lectin (AL) for various time periods in a serum- and growth factor-free medium and were subjected to various functional assays. Reactive oxygen species (ROS) levels were detected by using DCHFDA method. Cell fractionation was carried out using lectin-coupled paramagnetic beads. RESULTS: CD34+ cells incubated with the lectins for 10 days gave rise to a significantly higher number of colonies compared to the controls, indicating that all four lectins possessed the capacity to protect HSPCs in vitro. Comparative analyses showed that the protective ability of BL and GL was better than AL and DL and, therefore, further experiments were carried out with them. The output of long-term culture-initiating cell (LTC-IC) and extended LTC-IC assays indicated that both BL and GL protected primitive stem cells up to 30 days. The cells incubated with BL or GL showed a substantial reduction in the ROS levels, indicating that these lectins protect the HSPCs via antioxidant mechanisms. The mononuclear cell fraction isolated by lectin-coupled beads got enriched for primitive HSPCs, as reflected in the output of phenotypic and functional assays. CONCLUSION: The data show that both BL and GL protect the primitive HSPCs in vitro and may also serve as cost-effective HSPC enrichment tools.

A simple two-step culture system for the large-scale generation of mature and functional dendritic cells from umbilical cord blood CD34+ cells.

BACKGROUND: In vitro generated dendritic cells (DCs) are widely used as adjuvants in cancer immunotherapy. The major sources for DC generation are monocytes and CD34+ cells. CD34+-derived DCs are less frequently used in clinical applications because it requires complex generation methods. Here a simple method for the large-scale generation of mature functional DCs from umbilical cord blood-derived CD34+ cells is described. STUDY DESIGN AND METHODS: CD34+ cells were first expanded with a combination of early acting growth factors in a medium containing autologous plasma. In the second step the DC precursors were further either enriched by plastic adherence or sorted on a cell sorter and differentiated as DCs. DCs generated by both methods were compared for their morphology, phenotype, and different functional variables. RESULTS: This culture system provided a large-scale expansion of CD34+ cells giving a mean fold increase of 615. The majority of the expanded cells were interstitial DC precursors, that is, CD14+-positive cells. In vitro generated immature DCs could be matured into functional DCs by appropriate maturation stimuli. DCs generated by the plastic adherence method had a better cytokine profile and strong mixed leukocyte reaction compared to those generated by cell sorting. CONCLUSION: A two-step culture system provides a large-scale expansion of CD34+ cells with a preferential lineage commitment toward CD14+ cells. Enrichment of these precursors with a simple plastic adherence technique results in generation of large numbers of mature, functional DCs. This method of in vitro DC generation will have applications in cancer immunotherapy.

Prevention of apoptosis as a possible mechanism behind improved cryoprotection of hematopoietic cells by catalase and trehalose.

BACKGROUND: Our previous in vitro work has shown the usefulness of membrane stabilizers and antioxidants as additives in conventional freezing medium to freeze mouse and human hematopoietic cells. The present work was carried out using murine model to test the in vivo engraftment ability of mouse bone marrow frozen with (test cells) or without (control cells) addition of a combination of trehalose and catalase in the medium containing 10% dimethyl sulfoxide (DMSO). METHODS: Viability, nucleated cell recovery, and progenitor content of revived cells were measured. Freezing efficacy was tested by in vivo assays like colony forming unit-spleen (CFU-S), pre-CFU-S, and short-term engraftment of frozen marrow in irradiated mice. Long-term engraftment ability of frozen marrow was assessed using a Ly5.1-Ly 5.2 chimera model. Levels of apoptosis and reactive oxygen species (ROS) generated in revived cells were estimated. The former by Annnexin V, TUNEL, and DNA laddering and the latter by DCFH-DA probe. RESULTS: Our results show that the combination of catalase and trehalose with 10% DMSO improves freezing efficacy not only in terms of viability, cell recovery, and progenitor content but also by in vivo assays like CFU-S, pre-CFU-S, and short- and long-term engraftment. Both the level of apoptosis and ROS generation were considerably reduced in test set as compared to control set. CONCLUSIONS: We conclude that inclusion of a combination of trehalose and catalase in conventional freezing medium leads to enhanced engraftment potential of cryopreserved mouse bone marrow cells probably by preventing apoptotic cell death. Our observation using animal model may have significant clinical implications.

A combination of catalase and trehalose as additives to conventional freezing medium results in improved cryoprotection of human hematopoietic cells with reference to in vitro migration and adhesion properties.

BACKGROUND: Cryopreservation of hematopoietic cells from cord blood is an essential component in unrelated transplant settings. Cell damage during freezing is caused by multiple factors, of which membrane damage and oxygen free radical generation are two major factors. It was reported earlier that a combination of catalase and trehalose as additives in freezing medium affords better cryoprotection in terms of long-term culture assays. STUDY DESIGN AND METHODS: Mononuclear and CD34+ cells isolated from cord blood were used as a source of hematopoietic cells. KG1a cell line was used as a model system in adhesion assays. The cells were frozen in a programmable freezer and stored at -196 degrees C. Various homing-related assays were carried out on the frozen cells. RESULTS: Herein it is reported that these two additives afford better preservation of adhesion- and migration-related properties of the frozen cells. The test cells frozen with additives resulted in improved migration toward stromal-derived factor-1alpha and showed higher expression of its receptor CXCR4. Colony-forming unit assay of migrated test cells showed that these cells are early progenitors having capacity to give rise to all types of myeloid colonies. Test cells also show increased expression of FLT3R and improved responsiveness to FLT3 ligand, the homing-related cytokine. Adhesion to stroma and extracellular matrix was better in test cells as compared to control cells. CONCLUSION: The present data provide evidence that addition of catalase and trehalose to the conventional freezing medium preserves migration- and adhesion-related properties of the hematopoietic graft.

Supplementation of conventional freezing medium with a combination of catalase and trehalose results in better protection of surface molecules and functionality of hematopoietic cells.

Our previous studies had shown that a combination of the bio-antioxidant catalase and the membrane stabilizer trehalose in the conventional freezing mixture affords better cryoprotection to hematopoietic cells as judged by clonogenic assays. In the present investigation, we extended these studies using several parameters like responsiveness to growth factors, expression of growth factor receptors, adhesion assays, adhesion molecule expression, and long-term culture-forming ability. Cells were frozen with (test cells) or without additives (control cells) in the conventional medium containing 10% dimethylsulfoxide (DMSO). Experiments were done on mononuclear cells (MNC) from cord blood/fetal liver hematopoietic cells (CB/FL) and CD34(+) cells isolated from frozen MNC. Our results showed that the responsiveness of test cells to the two early-acting cytokines, viz. interleukin-3 (IL-3) and stem cell factor (SCF) in CFU assays was better than control cells as seen by higher colony formation at limiting concentrations of these cytokines. We, therefore, analyzed the expression of these two growth factor receptors by flow cytometry. We found that in cryopreserved test MNC, as well as CD34(+) cells isolated from them, the expression of both cytokine receptors was two- to three-fold higher than control MNC and CD34(+) cells isolated from them. Adhesion assays carried out with CB/FL-derived CD34(+) cells and KG1a cells showed significantly higher adherence of test cells to M210B4 than respective control cells. Cryopreserved test MNC as well as CD34(+) cells isolated from them showed increased expression of adhesion molecules like CD43, CD44, CD49d, and CD49e. On isolated CD34(+) cells and KG1a cells, there was a two- to three-fold increase in a double-positive population expressing CD34/L-selectin in test cells as compared to control cells. Long-term cultures (LTC) were set up with frozen MNC as well as with CD34(+) cells. Clonogenic cells from LTC were enumerated at the end of the fifth week. There was a significantly increased formation of CFU from test cells than from control cells, indicating better preservation of early progenitors in test cells. Our results suggest that use of a combination of catalase and trehalose as a supplement in the conventional freezing medium results in better protection of growth factor receptors, adhesion molecules, and functionality of hematopoietic cells, yielding a better graft quality.