Lalita Limaye

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.

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.

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.

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 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.

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.

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 TGFβ1 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.

Oral Administration of Insulin Receptor-Interacting Lectins Leads to an Enhancement in the Hematopoietic Stem and Progenitor Cell Pool of Mice

Lectins form an important constituent of our daily diet, and thus, it is essential that their effect(s) on various tissues be examined systematically in order to assess whether they are beneficial or detrimental to human health. We examined the effect of oral administration of two dietary lectins that were isolated from banana (BL) and garlic (GL)-two quite commonly consumed food items-on the hematopoiesis of mice. Balb/c mice were fed weekly with lectins and their marrow mononuclear cells (MNCs) were subjected to various hematopoietic stem/progenitor (HSPC)-specific phenotypic and functional assays. It was observed that the lectin-fed mice harbored a considerably increased HSPC pool in their marrow. Marrow-derived MNCs isolated from these lectin-fed mice gave rise to large-sized colony-forming unit-fibroblast (CFU-F) colonies indicating that the lectins had a salutary effect on the stromal compartment. The molecular mechanisms involved in the process were examined by using a stromal cell line model, M210B4. The lectins pulled down pro-insulin and insulin receptors in an immunoprecipitation experiment and activated extracellular signal-regulated kinase (ERK) signaling in the treated cells, in a manner comparable to insulin, both in terms of kinetics as well as extent. M210B4 cells incubated with BL, GL, or insulin showed reduced levels of reactive oxygen species, suggesting that perhaps the lectins protected the stem cell pool of mice by activating ERK signaling and reducing the oxidative stress in the niche. Our data suggest that these lectins may serve as micronutrients for therapeutic purposes in hematological deficiencies.

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.

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.