Nontoxic levels of cocaine exposure depresses multilineage hematopoiesis from human CD34 + HSPC

We first sought to determine if exposure to physiologically relevant concentrations of cocaine is toxic to human hematopoietic stem progenitor cells (HSPCs). Cocaine treatment levels and exposure time were used based on previous studies that determined on the basis of physiological effect on T cells without any toxicity risks22,26. CD34 + progenitors from fetal liver (FL) were incubated at varying timepoints with cocaine. Following treatment, a portion of the cells from both cocaine exposed and untreated cultures were examined by flow cytometry for cell death. Representative flow cytometry plots indicate no apparent differences in cell viability comparing untreated and cocaine treated cells at 24 and 48 hours post-exposure (Fig. 1a,b). Prior to cocaine exposure, cultures were approximately 85% viable while they were approximately 86% viable at 24 and 48 hours post drug exposure. Cultures were further examined for up to 10 days following exposure and monitored for evidence of toxicity(Fig. 1a,b). There were no evident differences in the viability of cultures that were exposed to cocaine compared to those that were not. These data indicate that cocaine is nontoxic to HSPCs in culture at a concentration of 10−8 M.

Figure 1 Cocaine is not immediately toxic to HSPCs. Representative live/dead dye exclusion flow cytometry viability assay of (A) cocaine treated and (B) untreated HSPC in culture for ten days. For each, top left panel: 0 hrs, top middle panel: 24 hrs, top right panel: 48 hours, bottom left panel: 5 days, bottom middle panel: 7 days, bottom right panel: 10 days. Full size image

Since cocaine had no apparent impact on cell viability, we then addresed whether cocaine exposure alters the hematopoietic potential of HSPCs derived from either FL, cord blood (CB), or adult bone marrow (BM). A portion of the cells from cocaine treated and untreated cultures were plated in complete methylcellulose and analyzed two weeks later for the development of hematopoietic colonies. In plates derived from HSPCs treated with cocaine, total colony number was severely reduced compared to untreated HSPCs (Fig. 2a,2b,2c). In FL derived cultures from three separate donors, cocaine treated cells gave rise to an average of 23 +/− 3 total colonies while untreated cells gave rise to an average of 87 +/− 17 total colonies. This difference is statistically significant by two tailed t-test with a P value <0.001. In CB derived cultures from three separate donors, cocaine treated cells gave rise to an average of 25 +/− 3 total colonies while untreated cells gave rise to an average of 93 +/− 17 total cells. This difference is statistically significant by two-tailed t-test with a p value <0.001. In BM derived cultures from three individual donors, cocaine treated progenitors gave rise to an average of 13 +/− 3 total colonies while untreated progenitors gave rise to an average of 44 +/− 5 total colonies. This difference is statistically significant by two-tailed t-test with a p value <0.001.

Figure 2 Nontoxic cocaine exposure impairs multilineage hematopoiesis. (A) Methylcellulose colony forming assay for untreated and cocaine treated HSPC derived from three different FL donors. Each mark represents the score on a sinlge plate and the bar marks the mean. (B) Methylcellulose colony forming assay for untreated and cocaine treated HSPC derived from three different CB donors. Each mark represents the score on a sinlge plate and the bar marks the mean. (C) Methylcellulose colony forming assay for untreated and cocaine treated HSPC derived from three different BM donors. Each mark represents the score on a sinlge plate and the bar marks the mean. (D) Phenotypic analysis of colonies that developed from FL derived HSPCs treated with cocaine. (E) Phenotypic analysis of colonies that developed from CB derived HSPCs treated with cocaine. (F) Phenotypic analysis of colonies that developed from BM derived HSPCs treated with cocaine. Red boxes/E erythroid colonies, blue boxes/G granulocyte colonies, black boxes/M macrophage colonies, green boxes/GM mixed granolocyte/macrophage colonies. Full size image

When colonies are analyzed by phenotype, development of all lineages are severely impaired in cultures derived from FL (fig. 2d), CB (fig. 2e) and BM (fig. 2e). Indeed, Table 1 shows that in almost every case derived from FL and CB, each type of colony from each donor, from both source tissue types, HSPCs that have been exposed to nonlethal levels of cocaine are impaired for development of all cell types. There is no apparent pattern of donor/colony types in which development is not impaired in a statistically significant fashion following drug exposure. All erythroid colonies from all but donor 3 showed that difference between the number of colonies that develop from untreated compared to cocaine treated progenitors. It is difficult to interpret the colony numbers of both the erythroid and the granulocyte/macrophage mixed cultures due to the fact that the numbers of these colony types in untreated cultures were quite low. However, there is strong significance in the differences in total colony number from this donor. BM derived cultures show similar results. While there were no differences in erythroid colonies that developed from cocaine treated compared to untreated progenitors, the development of almost all other myeloid colonies from all donors were suppressed in cultures derived from treated progenitors. Overall, these data indicate that cocaine exposure suppresses myeloid development from HSPCs derived from multiple tissues and types.

Table 1 Hematopoietic colonies derived from untreated or cocaine exposed CD34 + cells from either fetal liver, cord blood, or adult bone marrow. Donors 1–3 represent fetal liver, donors 4–6 represent cord blood and donors 7–9 represent adult bone marrow. Values are the Mean and SEM. E, erythroid, M, macrophage, G, granulocyte, GM, mixed granulocyte-macrophage colonies Full size table

The sigma-1 receptor is expressed in human CD34+ HSPCs

As previous work has implicated the sigma-1 receptor in mediating the cellular effects of cocaine22,26, we sought to determine if this receptor is present in human HSPCs. RT-PCR anaylsis for the sigma-1 receptor transcript in FL derived HSPCs from four separate donors indicate that it is indeed expressed in blood progenitors (Fig. 3a). The average of three RT-PCR reactions from donor 1 indicated 0.14 +/− 0.0078 copies of S1R per copy of GAPDH, while it was 0.17 +/− 0.0083 for donor 2, 0.11 +/− 0.013 for donor 3 and 0.14 +/− 0.0088 for donor 4.

Figure 3 The sigma-1 receptor is expressed in HSPCs and mediates cocaine's hematosuppressive effects. (A) RT-PCR analysis of sigma-1 receptor in FL derived HSPCs from four separate donors. S1R expression is graphed relative to GAPDH expression and the average of three replicate reactions is plotted. (B) Methylcellulose colony forming assays of FL derived HSPCs that were either untreated, cocaine treated, S1R block treated and cocaine + block treated. Each mark represents the score on a single plate and the bar marks the mean. (C) Representative images of hematopoietic colonies that developed from S1R-block treated (top) or untreated (bottom) HSPC. (D) Phenotypic analysis of colonies that developed from HSPCs that were either untreated, cocaine treated, S1R block treated and cocaine + block treated. Red boxes/E erythroid colonies, blue boxes/G granulocyte colonies, black boxes/M macrophage colonies, green boxes/GM mixed granolocyte/macrophage colonies. Full size image

S1R blockade alleviates cocaine-induced suppression of hematopoiesis

To elucidate the mechanism by which cocaine suppresses hematopoiesis, we expanded our cocaine exposure experiments to include blockade of the sigma-1 receptor. FL derived HSPCs from three separate donors were either left untreated or treated with cocaine, S1R blockade, or both for 48 hours and then plated in complete methylcellulose. Two weeks later, plates were scored for hematopoietic colony development. The total number of colonies that developed in cultures derived from cells that were treated with either S1R blockade or cocaine and S1R blockade were indiscernible while cultures that were treated with cocaine alone show significantly reduced total colony numbers (Fig. 3b). In this set of experiments, untreated HSPCs from all three donors gave rise to an average of 56 +/− 13 colonies per plate while those exposed to cocaine gave rise to only 12 +/− 2 colonies per plate. HSPCs that were treated with only the receptor block gave rise to 51 +/− 12 colonies and HSPCs that were treated with receptor block and cocaine gave rise to 51 +/− 13 colonies per plate. There were no significant differences in the total colony number that developed from untreated, receptor block treated, or receptor block and cocaine treated HSPCs.

Phenotypic analysis indicates that neither treatment with S1R block or S1R block and cocaine appears to alter hematopoietic lineage commitment (Fig. 3d). To confirm that treatment with the receptor block is not toxic to developing hematopoietic colonies, individual colonies from each of the treatment conditions were examined by microscopy (Fig. 3c). Table 2 shows that only cocaine exposure results in a statistically significant loss of developmental potential. Interestingly, macrophages were the only lineage that was not impaired with cocaine treatment. Granulocyte, macrophage and mixed granulocyte/macrophage colonies were clearly suppressed by cocaine exposure of HSPCs from all three donors. There were no statistically significant differences in the colony forming potential between untreated HSPC and those treated with just S1R blockade or both S1R blockade and cocaine. Together these data clearly show that the S1R can mediate changes in human hematopoiesis.