CD29 of human umbilical cord mesenchymal stem cells is required for expansion of CD34(+) cells

Abstract

Objectives: Human umbilical cord mesenchymal stem cells (hUCMSCs) play a critical role in expanding haematopoietic stem cells (HSCs) by providing the essential microenvironment for haematopoiesis. In this study, we sought to investigate whether CD29 of hUCMSCs would play a key role in the ability of hUCMSCs to help expand HSCs in vivo and in vitro.

Material and methods: To investigate whether CD29 of hUCMSCs would play a key role for the ability of hUCMSCs to expand HSCs, soluble anti-CD29 antibody was added to co-cultures of hUCMSCs and cord blood (CB) CD34(+) cells. It significantly blocked expansion of CB CD34(+) cells induced by hUCMSCs. Using CD29-deficient hUCMSCs models, long-term culture-initiating cell and non-obese diabetic/severe combined immunodeficient disease mouse repopulating cell assay, revealed that CB CD34(+) cells co-cultured with CD29-deficient hUCMSCs only retained the capacity of multipotent differentiation for 5 weeks at the most.

Results: Soluble anti-CD29 antibody significantly blocked expansion of CB CD34(+) cells induced by hUCMSCs. CB CD34(+) cells co-cultured with CD29-deficient hUCMSCs only retained the capacity of multipotent differentiation for 5 weeks at the most.

Conclusions: CB CD34(+) cells co-cultured with CD29-deficient hUCMSCs gave rise to all major haematopoietic lineages, but failed to engraft long term.

Figure 1

Ex vivo expansion of CB CD34 ⁺ cells with human umbilical cord mesenchymal stem cells ( hUCMSC s). (a) Total nucleate cells expansion after 14 days co‐culture with or without hUCMSCs, in the presence of cytokine cocktail. Data expressed as mean ± SE of mean (n = 5); *P < 0.05. (b) Flow cytometric analysis of CD29 expression in hUCMSCs. Blue histogram: isotype control; red histogram: specific antibody. Data shown are from one representative experiment of three reproducible experiments.

Figure 2

CD29 was required for HSC‐supporting activity of human umbilical cord mesenchymal stem cells ( hUCMSC s). (a) A total of 2.0 × 10⁴ CB CD34⁺ cells were co‐cultured with hUCMSCs in the presence of soluble mAb to CD29 or isotype mAb for 7 days, and harvested for flow cytometric analysis. Values indicate fold increase compared to initial number of cells. Data given as mean ± SD of three separate experiments. *P < 0.05, compared to cultures without antibody, or with isotype mAb. (b) Real‐time RT‐PCR was performed to evaluate expression level of CD29 in hUCMSCs after transduction with vectors for CD29 knockdown (KD1 or KD2), or control (CTRL). Data presented as ratio of CD29 levels in hUCMSCs, KD1 or KD2 to that in CTRL. Each reaction was performed in triplicate.

Figure 3

Effect of CD29 knockdown in human umbilical cord mesenchymal stem cells ( hUCMSC s) on long‐term culture‐initiating cell activity of CB CD34 ⁺ cells. Total of 1.0 × 10⁴ CB CD34⁺ cells were co‐cultured with KD1 or CTRL cells for 3–7 weeks, then subject to CFU assay. After 14–16 days culture, colonies, including BFU‐Es, CFU‐GMs, and CFU‐Mix, with greater than 50 cells were counted. Results expressed as mean ± SD (n = 5). *P < 0.05, compared between KD1 and CTRL cells (Student's t‐test).

Figure 4

Effect of CD29 knockdown in human umbilical cord mesenchymal stem cells ( hUCMSC s) on repopulation of CB CD34 ⁺ cells in NOD/SCID mice. (a) A total of 5.0 × 10⁴ CB CD34⁺ cells were co‐cultured with KD1 or CTRL for 4 weeks and then injected intravenously into sublethally irradiated NOD/SCID mice (n = 6 per group). Kinetic analysis of peripheral blood engraftment (Student's t‐test). (b) Level of total human cell engraftment. The mice were sacrificed 12 weeks after transplantation and mononuclear cells from bone marrow were analysed by flow cytometry. P = 0.026 compared between NOD/SCID mice injected with CD34⁺ cells co‐cultured with KD1 and those injected with CD34⁺ cells co‐cultured with CTRL. (c) Fraction of CD45⁺, erythroid and CD45⁺ CD34⁺ population among the engrafted human cells. *P < 0.05. (d) Flow cytometric analysis of human CB CD34⁺ cell repopulation in a representative primary NOD/SCID mouse after co‐culture with KD1 or CTRL cells. Mononuclear cells from bone marrow of engrafted NOD/SCID mice were examined for assessment of human CD45⁺ cells (R1) and erythroid cells including CD45⁻ CD36⁺ (R2) and CD36⁻ GPA⁺ (R3) population and CD45⁺ CD34⁺ cells (R4).