Human placenta is a potent hematopoietic niche containing hematopoietic stem and progenitor cells throughout development

Abstract

Hematopoietic stem cells (HSCs) are responsible for the life-long production of the blood system and are pivotal cells in hematologic transplantation therapies. During mouse and human development, the first HSCs are produced in the aorta-gonad-mesonephros region. Subsequent to this emergence, HSCs are found in other anatomical sites of the mouse conceptus. While the mouse placenta contains abundant HSCs at midgestation, little is known concerning whether HSCs or hematopoietic progenitors are present and supported in the human placenta during development. In this study we show, over a range of developmental times including term, that the human placenta contains hematopoietic progenitors and HSCs. Moreover, stromal cell lines generated from human placenta at several developmental time points are pericyte-like cells and support human hematopoiesis. Immunostaining of placenta sections during development localizes hematopoietic cells in close contact with pericytes/perivascular cells. Thus, the human placenta is a potent hematopoietic niche throughout development.

Figure 1

Human placenta contains hematopoietic progenitors throughout development. (A) Procedure for the isolation of cell populations from the human placenta. (B) Flow cytometric analyses of term blood and placenta. Cord blood cells, placental blood cells, cells recovered after extensive washes of the placental vasculature (Vessels PBS), from collagenase treatment of the placental vessels and subsequent collagenase treatment of the remaining placenta tissue were stained with anti-human (h)CD34 and CD38 antibodies and viable cells were analysed. Mean percentage ± SD (n=1–4) of relevant populations are indicated. (C) Clonogenic progenitors in term placenta were analysed in methylcellulose cultures. Frequency of total hematopoietic progenitors (CFU=colony forming unit) in the CD34⁺ cell fraction sorted from the different tissues. Sort purity for cord blood >96%, placenta blood >98%, vessel collagenase >93% and placenta collagenase >81%. Error bars display SEM (n=5). (D) Clonogenic progenitors in the sorted CD34⁺ (92–94% purity) and CD34⁻ (98–100% purity) cell fractions of early stage placentas were analysed in methylcellulose cultures. Frequencies of the different hematopoietic progenitor types (BFU-E, CFU-GM, CFU-Mix and the sum of these, Total CFU) in both CD34⁺ and CD34⁻ cell fractions sorted from placentas of gestational week 6, 9 and 15 are displayed. (E) Villus and (F) vasculature from 16 human placenta cryosections: CD34 (red), CD45 (green) and merged fluorescence are shown.

Figure 2

Long-term multilineage NOD-SCID hematopoietic repopulating cells are present in placenta throughout gestation. Human placenta cell engraftment was examined by (A) PCR for the human amelogenin gene (AMEL) or for the human chromosome 17 alpha-satellite sequence (h chr17) in blood (Bl), spleen (Sp), bone marrow (BM) and/or thymus (Th) and lymph node (LN) DNA isolated from cells of NOD-SCID mice transplanted with collagenase/dispase/pancreatin treated placenta tissue cells from the 6, 9 and 19 week (wk) gestation stages. 1.5×10⁶ of TC, 3×10⁶ of TCB, 3×10⁶ of TCA and 3×10⁶ of TC69A placenta cells were injected per mouse. TC, TCB, and TCA placentas were from male conceptuses and TC69A was from a female conceptus. TC, TCB, TCA and TC69A recipients were analysed respectively at 6, 10, 11 and 7 weeks post-transplantation. (B) Flow cytometric multilineage analyses of blood, bone marrow (BM) and spleen cells isolated from NOD-SCID mice 10 weeks after injection of 3×10⁶ cells from collagenase/dispase/pancreatin treated TCB placenta tissue. Cells were stained with anti-mouse (m) CD45 and anti-human (h) CD34, CD38, CD45, CD19 and CD15 antibodies and analysed in the viable population. Number of events analysed were 3×10⁵ for blood and 9×10⁴ for BM and spleen. Percentages of gated populations are indicated. (C) Frequencies of the different hematopoietic progenitor types (BFU-E, CFU-G, CFU-M, CFU-Mix) present in the total BM isolated from the TCB reconstituted NOD-SCID recipient shown in panels A and B. (D) PCR analysis for the amelogenin gene was performed on each colony type and on a pool of colonies (CFU pool) harvested from the culture experiments in panel C. The presence of AMELY fragment reveals their fetal origin. (E) STR profiling of DNA from the spleen and BM of the NOD-SCID recipient transplanted with TC69A (female) placenta tissue cells. TC69A embryo DNA (female) served as the control for fetal-derived cells. STR alleles are designated as numbers of polymorphic repeats.

Figure 3

Long-term multilineage NOD-SCID hematopoietic repopulating potential of full term placenta cells. Flow cytometric multilineage analyses of blood, bone marrow (BM) and spleen cells isolated from NOD-SCID mice repopulated 5 months after injection of term placenta cells. (A) 20×10⁶ cells from collagenase treated placenta tissue (tP2); (B) 7×10⁶ cells from collagenase treated placental vessels (tP2); and (C) 10×10⁶ cells from collagenase/dispase/pancreatin treated placenta tissue (tP3). Cells are stained with anti-mouse (m) CD45 and/or anti-human (h) CD45, CD19, CD15, CD34 and CD38 antibodies and analysed in the viable population. Number of events analysed was 3×10⁴ for all tissues in panels A and B, and 2×10⁵ for blood and 1×10⁵ for BM and spleen in panel C. Percentages of gated and quadrant populations are indicated. (D) To verify the fetal (male) origin of the engraftment, PCR for the amelogenin gene was performed on blood (Bl), spleen (Sp), bone marrow (BM), thymus (Th) and lymph node (LN) DNA isolated from cells of the reconstituted recipients described in panels A, B and C. Control female (XX) cell DNA produces a single product (AMELX at 106 bp), whereas control male (XY) DNA produces two products (AMELY at 112 bp and AMELX).

Figure 4

Pericyte marker expression on human placenta stromal cell lines and human placenta tissue. (A) Histogram of flow cytometric analysis for NG2 and CD146 expression on H92.1 placenta stromal cell line is shown. Immunostained cryosections from 16 week human placenta co-stained for (B) CD146 (red) and α-SMA (green) (20× lens) or (C) NG2 (red) and α-SMA (green) (10× lens). Single and merged fluorescence are shown. MV=microvessels; C=capillaries; LV=large vessel.