Mesenchymal stem cells are short-lived and do not migrate beyond the lungs after intravenous infusion

Abstract

Mesenchymal stem cells (MSC) are under investigation as a therapy for a variety of disorders. Although animal models show long term regenerative and immunomodulatory effects of MSC, the fate of MSC after infusion remains to be elucidated. In the present study the localization and viability of MSC was examined by isolation and re-culture of intravenously infused MSC. C57BL/6 MSC (500,000) constitutively expressing DsRed-fluorescent protein and radioactively labeled with Cr-51 were infused via the tail vein in wild-type C57BL/6 mice. After 5 min, 1, 24, or 72 h, mice were sacrificed and blood, lungs, liver, spleen, kidneys, and bone marrow removed. One hour after MSC infusion the majority of Cr-51 was found in the lungs, whereas after 24 h Cr-51 was mainly found in the liver. Tissue cultures demonstrated that viable donor MSC were present in the lungs up to 24 h after infusion, after which they disappeared. No viable MSC were found in the other organs examined at any time. The induction of ischemia-reperfusion injury in the liver did not trigger the migration of viable MSC to the liver. These results demonstrate that MSC are short-lived after i.v. infusion and that viable MSC do not pass the lungs. Cell debris may be transported to the liver. Long term immunomodulatory and regenerative effects of infused MSC must therefore be mediated via other cell types.

Keywords: infusion; liver; localization; lung; mesenchymal stem cell; survival.

Figure 1

DsRed-MSC are phenotypically and functionally comparable to wild-type MSC. (A) Fluorescence microscopy of plastic adherent DsRed-MSC in culture shows bright red fluorescence. (B) Flow cytometric analysis demonstrates that DsRed-MSC are negative for the expression of CD34, CD11b, CD11c, and CD117, show weak expression of MHC class I, and are positive for Sca-1, CD44, and RFP (DsRed). (C) DsRed-MSC are capable of differentiating into adipocytes, as demonstrated by staining of lipid vesicles by Oil-Red-O (left), while remaining red fluorescent (right). (D) DsRed-MSC are capable of differentiating into osteoblasts, indicated by positive silver nitrate staining for calcium deposits (left), while remaining red fluorescent (right). (E and F) DsRed-MSC suppress ConA induced proliferation of CD4 positive and negative T cells efficiently, determined by CFSE dilution on day 3 (solid line: ConA stimulated T cells, gray shaded curve: ConA stimulated T cells + MSC, dotted line: non-stimulated T cells). DsRed-MSC suppressed the proliferation of syngeneic C57BL/6 responder T cells (E) as well as allogeneic Balb/c responder T cells (F). Representative data of 3 experiments shown.

Figure 2

Distribution of Cr-51 radioactivity after infusion of Cr-51 labeled DsRed-MSC. (A) One hour after intravenous injection, more than 60% of total radioactivity is located in the lungs and approximately 10% in the liver. Other tissues contain only residual radioactivity. After 24 h, the majority of radioactivity is located in the liver. (B) As in (A), but absolute counts shown. Dashed line indicates background radioactivity. Cervical, axillary, inguinal, hepatic, para-aortal, and mesenteric lymph nodes were collected. Average data of 2 experiments is shown.

Figure 3

Viable MSC are detected in lung tissue only after intravenous infusion. (A) Brightfield, immunofluorescent, and merged microscopic images show two DsRed-MSC in a two days-old culture of lung tissue established 5 min after MSC infusion. (B) After 7 days of culture clusters of DsRed-MSC are seen. (C) Flow cytometric analysis of cultures established from lung tissue 5 min after DsRed-MSC infusion cultured for 7 days revealed a distinct population of CD44⁺ DsRed-MSC (middle plot), while in cultures of PBS treated animals no DsRed-MSC were present (left plot). Mono-cultures of DsRed-MSC served as gating control (right plot). Five animals were included per group. Representative data shown.