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Phenotypic, Morphological and Adhesive Differences of Human Hematopoietic Progenitor Cells Cultured on Murine versus Human Mesenchymal Stromal Cells.

Reichert D, Friedrichs J, Ritter S, Käubler T, Werner C, Bornhäuser M, Corbeil D - Sci Rep (2015)

Bottom Line: The number of migrating HSPCs was also reduced on murine cells suggesting that MSC adhesion influences cellular polarization of HSPC.We found threefold higher detachment forces of human HSPCs from murine MSCs compared to human ones.Our observations highlight phenotypic, morphological and adhesive differences of human HSPCs when cultured on murine or human MSCs, which raise some caution in data interpretation when xenogenic transplantation models are used.

View Article: PubMed Central - PubMed

Affiliation: Tissue Engineering Laboratories (BIOTEC), Technische Universität Dresden, 01307 Dresden, Germany.

ABSTRACT
Xenogenic transplantation models have been developed to study human hematopoiesis in immunocompromised murine recipients. They still have limitations and therefore it is important to delineate all players within the bone marrow that could account for species-specific differences. Here, we evaluated the proliferative capacity, morphological and physical characteristics of human CD34(+) hematopoietic stem and progenitor cells (HSPCs) after co-culture on murine or human bone marrow-derived mesenchymal stromal cells (MSCs). After seven days, human CD34(+)CD133(-) HSPCs expanded to similar extents on both feeder layers while cellular subsets comprising primitive CD34(+)CD133(+) and CD133(+)CD34(-) phenotypes are reduced fivefold on murine MSCs. The number of migrating HSPCs was also reduced on murine cells suggesting that MSC adhesion influences cellular polarization of HSPC. We used atomic force microscopy-based single-cell force spectroscopy to quantify their adhesive interactions. We found threefold higher detachment forces of human HSPCs from murine MSCs compared to human ones. This difference is related to the N-cadherin expression level on murine MSCs since its knockdown abolished their differential adhesion properties with human HSPCs. Our observations highlight phenotypic, morphological and adhesive differences of human HSPCs when cultured on murine or human MSCs, which raise some caution in data interpretation when xenogenic transplantation models are used.

No MeSH data available.


Related in: MedlinePlus

The relative amount of human HSPCs exhibiting a migrating morphology is reduced on murine MSCs in comparison to human ones.(a) Phase contrast pictures of human (h) HSPCs growing on either h (top panels) or murine (m; bottom panels) MSCs for 7 days. The absolute amounts of migrating and non-migrating hematopoietic cells, which have an elongated (green) or spherical (red) morphology, respectively, observed in each frames are indicated. Left panels show higher magnification views of areas indicated in the corresponding images on the right. (b,c) Numbers of hematopoietic cells exhibiting morphologies characteristic of non-migrating (NM, red) and migrating (M, green) cells, respectively (see Fig. 2), were counted after 24 hours and 7 days of co-culture on MSCs (b). Numbers CD133+ hHSPCs with a given morphology after 7 days are indicated (c). Three to four experiments were performed using CD34+ hHSPCs isolated from different donors, and at least 400 (b) or 100–200 (c) cells were analyzed per condition. Three asterisks indicate a significant difference as calculated by two-tailed paired student’s t-test (p < 0.001). N.s., not significant (p ≥ 0.05). Scale bars: 10 μm.
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f3: The relative amount of human HSPCs exhibiting a migrating morphology is reduced on murine MSCs in comparison to human ones.(a) Phase contrast pictures of human (h) HSPCs growing on either h (top panels) or murine (m; bottom panels) MSCs for 7 days. The absolute amounts of migrating and non-migrating hematopoietic cells, which have an elongated (green) or spherical (red) morphology, respectively, observed in each frames are indicated. Left panels show higher magnification views of areas indicated in the corresponding images on the right. (b,c) Numbers of hematopoietic cells exhibiting morphologies characteristic of non-migrating (NM, red) and migrating (M, green) cells, respectively (see Fig. 2), were counted after 24 hours and 7 days of co-culture on MSCs (b). Numbers CD133+ hHSPCs with a given morphology after 7 days are indicated (c). Three to four experiments were performed using CD34+ hHSPCs isolated from different donors, and at least 400 (b) or 100–200 (c) cells were analyzed per condition. Three asterisks indicate a significant difference as calculated by two-tailed paired student’s t-test (p < 0.001). N.s., not significant (p ≥ 0.05). Scale bars: 10 μm.

Mentions: Their migratory behavior was recorded using time-lapse video and cellular tracking (Fig. 2b). After 24 hours or 7 days of co-culture, the average migration distances of hematopoietic cells are similar independent of the species origin of the MSCs (Fig. 2c,d). However, we noticed during video recording that numerous hematopoietic cells were not moving, but maintain a spherical morphology typical of non-migrating cells (Fig. 3a). This prompted us to quantify the ratio of migrating and non-migrating cells. Surprisingly, the proportion of cells harboring a migratory morphology is significantly reduced when they were cultured for 7 days on mMSCs in comparison to their human counterparts (Fig. 3b). Such difference is not observed within the first 24 hours of cultivation (Fig. 3b). The presence (or absence) of CD133 did not change the ratio of migrating versus non-migrating cells (Fig. 3c), as previously reported30, indicating that other factors in these cell-based systems influence the hematopoietic cell behavior. Thus, our data suggest that hHSPCs are migrating similarly on both m and hMSCs, but the proportion of migrating cells is significantly reduced on murine feeder cells.


Phenotypic, Morphological and Adhesive Differences of Human Hematopoietic Progenitor Cells Cultured on Murine versus Human Mesenchymal Stromal Cells.

Reichert D, Friedrichs J, Ritter S, Käubler T, Werner C, Bornhäuser M, Corbeil D - Sci Rep (2015)

The relative amount of human HSPCs exhibiting a migrating morphology is reduced on murine MSCs in comparison to human ones.(a) Phase contrast pictures of human (h) HSPCs growing on either h (top panels) or murine (m; bottom panels) MSCs for 7 days. The absolute amounts of migrating and non-migrating hematopoietic cells, which have an elongated (green) or spherical (red) morphology, respectively, observed in each frames are indicated. Left panels show higher magnification views of areas indicated in the corresponding images on the right. (b,c) Numbers of hematopoietic cells exhibiting morphologies characteristic of non-migrating (NM, red) and migrating (M, green) cells, respectively (see Fig. 2), were counted after 24 hours and 7 days of co-culture on MSCs (b). Numbers CD133+ hHSPCs with a given morphology after 7 days are indicated (c). Three to four experiments were performed using CD34+ hHSPCs isolated from different donors, and at least 400 (b) or 100–200 (c) cells were analyzed per condition. Three asterisks indicate a significant difference as calculated by two-tailed paired student’s t-test (p < 0.001). N.s., not significant (p ≥ 0.05). Scale bars: 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4620509&req=5

f3: The relative amount of human HSPCs exhibiting a migrating morphology is reduced on murine MSCs in comparison to human ones.(a) Phase contrast pictures of human (h) HSPCs growing on either h (top panels) or murine (m; bottom panels) MSCs for 7 days. The absolute amounts of migrating and non-migrating hematopoietic cells, which have an elongated (green) or spherical (red) morphology, respectively, observed in each frames are indicated. Left panels show higher magnification views of areas indicated in the corresponding images on the right. (b,c) Numbers of hematopoietic cells exhibiting morphologies characteristic of non-migrating (NM, red) and migrating (M, green) cells, respectively (see Fig. 2), were counted after 24 hours and 7 days of co-culture on MSCs (b). Numbers CD133+ hHSPCs with a given morphology after 7 days are indicated (c). Three to four experiments were performed using CD34+ hHSPCs isolated from different donors, and at least 400 (b) or 100–200 (c) cells were analyzed per condition. Three asterisks indicate a significant difference as calculated by two-tailed paired student’s t-test (p < 0.001). N.s., not significant (p ≥ 0.05). Scale bars: 10 μm.
Mentions: Their migratory behavior was recorded using time-lapse video and cellular tracking (Fig. 2b). After 24 hours or 7 days of co-culture, the average migration distances of hematopoietic cells are similar independent of the species origin of the MSCs (Fig. 2c,d). However, we noticed during video recording that numerous hematopoietic cells were not moving, but maintain a spherical morphology typical of non-migrating cells (Fig. 3a). This prompted us to quantify the ratio of migrating and non-migrating cells. Surprisingly, the proportion of cells harboring a migratory morphology is significantly reduced when they were cultured for 7 days on mMSCs in comparison to their human counterparts (Fig. 3b). Such difference is not observed within the first 24 hours of cultivation (Fig. 3b). The presence (or absence) of CD133 did not change the ratio of migrating versus non-migrating cells (Fig. 3c), as previously reported30, indicating that other factors in these cell-based systems influence the hematopoietic cell behavior. Thus, our data suggest that hHSPCs are migrating similarly on both m and hMSCs, but the proportion of migrating cells is significantly reduced on murine feeder cells.

Bottom Line: The number of migrating HSPCs was also reduced on murine cells suggesting that MSC adhesion influences cellular polarization of HSPC.We found threefold higher detachment forces of human HSPCs from murine MSCs compared to human ones.Our observations highlight phenotypic, morphological and adhesive differences of human HSPCs when cultured on murine or human MSCs, which raise some caution in data interpretation when xenogenic transplantation models are used.

View Article: PubMed Central - PubMed

Affiliation: Tissue Engineering Laboratories (BIOTEC), Technische Universität Dresden, 01307 Dresden, Germany.

ABSTRACT
Xenogenic transplantation models have been developed to study human hematopoiesis in immunocompromised murine recipients. They still have limitations and therefore it is important to delineate all players within the bone marrow that could account for species-specific differences. Here, we evaluated the proliferative capacity, morphological and physical characteristics of human CD34(+) hematopoietic stem and progenitor cells (HSPCs) after co-culture on murine or human bone marrow-derived mesenchymal stromal cells (MSCs). After seven days, human CD34(+)CD133(-) HSPCs expanded to similar extents on both feeder layers while cellular subsets comprising primitive CD34(+)CD133(+) and CD133(+)CD34(-) phenotypes are reduced fivefold on murine MSCs. The number of migrating HSPCs was also reduced on murine cells suggesting that MSC adhesion influences cellular polarization of HSPC. We used atomic force microscopy-based single-cell force spectroscopy to quantify their adhesive interactions. We found threefold higher detachment forces of human HSPCs from murine MSCs compared to human ones. This difference is related to the N-cadherin expression level on murine MSCs since its knockdown abolished their differential adhesion properties with human HSPCs. Our observations highlight phenotypic, morphological and adhesive differences of human HSPCs when cultured on murine or human MSCs, which raise some caution in data interpretation when xenogenic transplantation models are used.

No MeSH data available.


Related in: MedlinePlus