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 homingrelated 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

Conditioned Medium from Placental Mesenchymal Stem Cells Reduces Oxidative Stress during the Cryopreservation of Ex Vivo Expanded Umbilical Cord Blood Cells.

The limited cell dose in umbilical cord blood (UCB) necessitates ex vivo expansion of UCB. Further, the effective cryopreservation of these expanded cells is important in widening their use in the clinics. During cryopreservation, cells experience oxidative stress due to the generation of reactive oxygen species (ROS). Conditioned medium from mesenchymal stem cells (MSCs-CM) has been shown to alleviate the oxidative stress during wound healing, Alzheimer's disease and ischemic disease. This premise prompted us to investigate the influence of MSCs-CM during cryopreservation of expanded UCB cells.CM-was collected from cord/placental MSCs(C-MSCs-CM, P-MSC-CM). UCB CD34+cells were expanded as suspension cultures in serum free medium containing cytokines for 10 days. Cells were frozen with/without C-MSCs-CM and or P-MSCs-CM in the conventional freezing medium containing 20%FCS +10%DMSO using a programmable freezer and stored in liquid nitrogen. Upon revival, cells frozen with MSCs-CM were found to be superior to cells frozen in conventional medium in terms of viability, CD34+content and clonogenecity. Priming of revived cells for 48 hrs with MSCs-CM further improved their transplantation ability, as compared to those cultured without MSCs-CM. P-MSCs-CM radically reduced the oxidative stress in cryopreserved cells, resulting in better post thaw functionality of CD34+ cells than with C-MSCs-CM. The observed cryoprotective effect of MSCs-CM was primarily due to anti-oxidative and anti-apoptotic properties of the MSCs-CM and not because of the exosomes secreted by them.Our data suggest that MSCs-CM can serve as a valuable additive to the freezing or the priming medium for expanded UCB cells, which would increase their clinical applicability

Additional file 1: Figure S1. of Differential ability of MSCs isolated from placenta and cord as feeders for supporting ex vivo expansion of umbilical cord blood derived CD34+ cells

BM-MSCs and P-MSCs had similar potential for expansion of CD34+ cells. a BM-MSCs have fibroblastic morphology, can be differentiated into osteoblasts, adipocytes and chondrocytes. b BM-MSCs were shown to exhibit expression of CD44, CD73, CD105, and CD90 and found not to express CD34, CD45, CD14, CD19, CD11b, and andHLA-DR. c BM-MSCs had robust expansion of total nucleated cells, CD34+CD38−, and CD133+ cells. d CD34+ cells expanded on BM-MSCs had superior clonogenic ability. Data are represented as mean ± standard deviation from four different samples. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. (TIFF 5033 kb

Actin dynamics of expanded HSPCs.

<p>(A) The expanded control and zVADfmk/zLLYfmk HSPCs were assessed for the polymerized actin (F actin) using fluorescein conjugated phalloidin. The presence of zVADfmk and zLLYfmk enhanced the of F actin fluorescence intensity compared to the control. Data are represented as mean ± standard deviation of four experiments, *<i>p</i>≤0.05. (B) Actin polymerization assay done by exposing the control and test HSPCs to SDF1α for different time intervals showed an early response and higher polymerization of actin at all time points analyzed in the zVADfmk/zLLYfmk sets. Data are represented as mean ± standard deviation of three experiments. Statistical analysis was always made between control vs. zVADfmk/zLLYfmk at all time points. *<i>p</i>≤0.05, **<i>p<</i>0.01. (C) The migrated population from the zVADfmk/zLLYfmk HSPCs showed a typical polarized morphology with a prominent localization of F actin towards the leading edge of the cells (white arrow heads & insets) where as in control cells a homogeneous distribution of F actin was seen without much visible cell polarization, green – F actin, bar = 10 µm. (D) The expression of RhoGTPase members RhoA, Rac1 and Cdc42 was found to be increased in the zVADfmk/zLLYfmk HSPCs, green –RhoA, Rac1 and Cdc42, bar = 10 µm (E) Quantitative real time PCR analysis showing a significant up regulation in the gene expression of RhoA upto (3.2 fold) and Cdc42 (up to 2.5 fold) in the zVADfmk/zLLYfmk expanded HSPCs compared to the control, whereas the Rac1 showed a modest increase in those sets. Data are represented as mean ± standard deviation of three experiments, *<i>p</i>≤0.05, **<i>p<</i>0.01.</p

Higher expression of adhesion molecules in the expanded HSPCs.

<p>(A) & (B & C) Flow cytometry analyses of HSPCs after staining for CD34 vs CD62L, CD34 vs CD54 and CD34 vs CD44 antibodies demonstrate higher positive population of these adhesion molecules on CD34⁺ progenitor cells. (D) Alternatively multi colour flow cytometry analyses showed that zVADfmk/zLLYfmk enhanced the presence of CD62L, CD44 and CD54 on CD34⁺CD38− subsets as well. Data are represented as mean percentage ± standard deviation of four experiments, *<i>p</i><0.05, **<i>p</i><0.01.</p

<i>In vitro</i> migration of expanded CB HSPCs.

<p>(A) The presence of zVADfmk/zLLYfmk during <i>ex vivo</i> expansion significantly increased the chemotaxis of HSPCs towards SDF1α compared to the cytokine-expanded (control) counterpart. Data are represented as mean ± standard deviation of four experiments, *<i>p</i>≤0.05, **<i>p</i>≤0.01. (B) The migration of expanded HSPCs towards SDF1α was reduced when a neutralizing antibody to CXCR4 was used, indicating the direct role of CXCR4 in maintaining the chemotactic responses. Data are represented as mean ± standard deviation of three experiments. (C) The colony forming assay of the migrated population showed a higher clonogenicity, especially the presence of primitive progenitors in terms of CFU mix/GEMM colonies in the zVADfmk/zLLYfmk cultures. Data obtained from three experiments are represented as mean ± standard deviation, *<i>p</i>≤0.05, **<i>p</i><0.01.</p