Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 May 29;9(6):1350.
doi: 10.3390/cells9061350.

Generation of Multipotent Stem Cells from Adult Human Peripheral Blood Following the Treatment with Platelet-Derived Mitochondria

Haibo Yu  1 , Wei Hu  1 , Xiang Song  1 , Yong Zhao  1
Affiliations

Generation of Multipotent Stem Cells from Adult Human Peripheral Blood Following the Treatment with Platelet-Derived Mitochondria

Haibo Yu et al. Cells. .

Abstract

Autologous stem cells are highly preferred for cellular therapy to treat human diseases. Mitochondria are organelles normally located in cytoplasm. Our recent studies demonstrated the differentiation of adult peripheral blood-derived insulin-producing cells (designated PB-IPC) into hematopoietic-like cells after the treatment with platelet-derived mitochondria. To further explore the molecular mechanism and their therapeutic potentials, through confocal and electron microscopy, we found that mitochondria enter cells and directly penetrate the nucleus of PB-IPC after the treatment with platelet-derived mitochondria, where they can produce profound epigenetic changes as demonstrated by RNA-seq and PCR array. Ex vivo functional studies established that mitochondrion-induced PB-IPC (miPB-IPC) can give rise to retinal pigment epithelium (RPE) cells and neuronal cells in the presence of different inducers. Further colony analysis highlighted the multipotent capability of the differentiation of PB-IPC into three-germ layer-derived cells. Therefore, these data indicate a novel function of mitochondria in cellular reprogramming, leading to the generation of autologous multipotent stem cells for clinical applications.

Keywords: PB-IPC; cell reprogramming; differentiation; mitochondria; multipotent stem cells; platelets.

PubMed Disclaimer

Conflict of interest statement

Dr. Zhao is a founder of Tianhe Stem Cell Biotechnology Inc. Dr. Zhao is an inventor of Stem Cell Educator technology and an inventor for technologies on platelets and mitochondria that have been submitted for patent applications (US 15/688 464, US 15/688 498). This work has been submitted for a provisional patent application. Y.Z, H.Y, W.H. and X.S. were listed as inventors.

Figures

Figure 1
Figure 1
Characterization of peripheral blood-derived insulin-producing cells (PB-IPC) from adult peripheral blood with islet β-cell-related markers. (A) Characterization of PB-IPC. Peripheral blood-derived mononuclear cells (PBMC) were plated in Petri dishes in the presence of serum-free culture medium. After attachment overnight (12 hrs), PB-IPC were isolated by removing all floating cells and debris. The gated Lin1-CD34- cells express CD45, SOX2, CD45RO, and CCR7. Representative data from eight preparations. (B) Phenotype of PB-IPC, with low expression of CD117, but no expression of CD4, CD8, CD19, CD34, CD38, CD41, CD42a, and CD66b. Isotype-matched IgGs served as controls (n = 8). (C) Apoptosis (Annexin V+) and necrosis (7-AAD+) of blood monocytes after 24-h culture in non-tissue culture-treated Petri dishes (n = 5). (D) Analysis of cell cycles in the freshly-isolated PB-IPC after overnight attachment by flow cytometry with propidium iodide (PI) staining (n = 5). (E) Real time PCR analysis of pancreatic islet β-cell-related markers in PB-IPC isolated from healthy donors (n = 5). Freshly isolated human islets served as positive controls. (F) Flow cytometry for islet β-cell-related transcription factor MAFA and an insulin by-product C-peptide by double immunostaining (n = 5). (G) Flow cytometry for determining PB-IPC’s phenotype after overnight attachment. Representative data from four preparations. FITC-conjugated anti-human lineage cocktail 1 (Lin1) (CD3, CD14, CD16, CD19, CD20, CD56) was applied to eliminate the known cell lineages such as T cells, monocytes/macrophages, granulocytes, B cells, and natural killer (NK) cells. Anti-human leukocyte common antigen CD45 mAb was used to remove the red blood cells (RBC) and platelets’ contamination during data analysis. MAFA (transcription factor) and GLUT2 (β cell surface marker) were utilized to determine the islet β cell-associated phenotype in PB-IPC. (H) Fluorescence microscopy shows a GFP+ cell among PBMC of an insulin promotor 1-GFP-transgenic mouse (n = 3). A GFP-positive mouse islet served as positive control.
Figure 2
Figure 2
Differentiation of mitochondrion-induced PB-IPC (miPB-IPC) into retinal pigment epithelium (RPE) cells. (A) Phase contrast images show the differentiation of miPB-IPC into RPE cells with cellular pigmentation and processes at varied lengths (n = 5). (B) Immunostaining of differentiated RPE cells with RPE-specific markers (n = 3). The human primary RPE cells served as positive controls. Mouse IgG and rabbit IgG merged with nuclear DAPI (blue) staining served as negative controls (inserts). Untreated miPB-IPC served as negative control (middle panel). (C) Phagocytosis of fluorescence beads by differentiated RPE cells (n = 3). (D) Flow cytometry analysis of CD36 expression on differentiated RPE cells (red) and untreated cells (blue) (n = 3). Isotype-matched IgG served as negative control (grey).
Figure 3
Figure 3
Differentiation of miPB-IPC into neuronal cells. (A) Phase contrast images show the differentiation of miPB-IPC into neuronal cells (n = 5). (B) Immunostaining of differentiated neuronal cells with neuron-specific markers synapsin I (red) and tyrosine hydroxylase (green) (n = 3). IgG staining served as negative control (inserts). Untreated miPB-IPC served as negative control (top panel).
Figure 4
Figure 4
Clonal analysis of miPB-IPC and testing for tumor formation of miPB-IPC. (A) Colony formation of miPB-IPC with different sizes in regular miPB-IPC cell cultures (n = 5). (B) Potential difference in colony formation between miPB-IPC relative to untreated PB-IPC. Increase the colony formation in miPB-IPC relative to untreated PB-IPC. The colony formation of miPB-IPC with different sizes at 2-month culture in 24-well plate. The miPB-IPC were initially cultured with the serum-free NutriStem® hPSC XF culture medium (Corning) at 1 × 104 cells/mL/well in 24-well tissue culture plates, at 37 °C in 8% CO2 culture condition. Data are presented as mean ± SD from five preparations. (C) Phenotypic analysis of single colony-derived cells, retaining the PB-IPC’s markers CD34-CD45+SOX2+CD45RO+CCR7+ (n = 5). (D) Clonal analysis. A single colony was dispersed and inoculated into 96-well plates, treated wells with different lineage-specific inducers for differentiations, including macrophages (left, phagocytosis of green fluorescence beads, n = 3), RPE cells (middle, n = 6), and neural cells (right, n = 9). (E) Gaining weight in miPB-IPC-transplanted mice, without tumor formation. The miPB-IPC at the dose of 2 × 107 cells/mouse were inoculated (s.c., right lower flank) in NOD-scid IL-2Rγnull mice (n = 3). Injection of equal volume of physiological saline on the left lower flank served as control. (F) Colony analysis with three-germ layer-associated markers such as a neuronal marker synapsin I for ectoderm, the islet β cell marker insulin for endoderm, and a macrophage marker CD11b for mesoderm. IgGs served as negative controls (top panel). Representative images were from one of eight colonies for miPB-IPC group (bottom panel) and five colonies for control PB-IPC (middle panel). (G) Colony analysis with additional three-germ layer-associated markers such as a neuronal marker beta III tubulin (Tuj1) for ectoderm, the liver cell marker alpha-fetoprotein (AFP) for endoderm, and smooth muscle actin (SMA) for mesoderm. IgGs served as negative controls (top panel). Representative images were from one of seven colonies for miPB-IPC group (bottom panel) and five colonies for control PB-IPC (middle panel).
Figure 4
Figure 4
Clonal analysis of miPB-IPC and testing for tumor formation of miPB-IPC. (A) Colony formation of miPB-IPC with different sizes in regular miPB-IPC cell cultures (n = 5). (B) Potential difference in colony formation between miPB-IPC relative to untreated PB-IPC. Increase the colony formation in miPB-IPC relative to untreated PB-IPC. The colony formation of miPB-IPC with different sizes at 2-month culture in 24-well plate. The miPB-IPC were initially cultured with the serum-free NutriStem® hPSC XF culture medium (Corning) at 1 × 104 cells/mL/well in 24-well tissue culture plates, at 37 °C in 8% CO2 culture condition. Data are presented as mean ± SD from five preparations. (C) Phenotypic analysis of single colony-derived cells, retaining the PB-IPC’s markers CD34-CD45+SOX2+CD45RO+CCR7+ (n = 5). (D) Clonal analysis. A single colony was dispersed and inoculated into 96-well plates, treated wells with different lineage-specific inducers for differentiations, including macrophages (left, phagocytosis of green fluorescence beads, n = 3), RPE cells (middle, n = 6), and neural cells (right, n = 9). (E) Gaining weight in miPB-IPC-transplanted mice, without tumor formation. The miPB-IPC at the dose of 2 × 107 cells/mouse were inoculated (s.c., right lower flank) in NOD-scid IL-2Rγnull mice (n = 3). Injection of equal volume of physiological saline on the left lower flank served as control. (F) Colony analysis with three-germ layer-associated markers such as a neuronal marker synapsin I for ectoderm, the islet β cell marker insulin for endoderm, and a macrophage marker CD11b for mesoderm. IgGs served as negative controls (top panel). Representative images were from one of eight colonies for miPB-IPC group (bottom panel) and five colonies for control PB-IPC (middle panel). (G) Colony analysis with additional three-germ layer-associated markers such as a neuronal marker beta III tubulin (Tuj1) for ectoderm, the liver cell marker alpha-fetoprotein (AFP) for endoderm, and smooth muscle actin (SMA) for mesoderm. IgGs served as negative controls (top panel). Representative images were from one of seven colonies for miPB-IPC group (bottom panel) and five colonies for control PB-IPC (middle panel).
Figure 5
Figure 5
Penetration of mitochondria into nuclei of PB-IPC. (A) Transmission electron microscopy demonstrates a mitochondrion (M) crossing the nuclear membrane of a mitochondrion-treated PB-IPC. (B) A mitochondrion located inside the nuclear matrix, and close to the nucleolus with a morphologically-similar mitochondrion (indicated by red arrow) in the cytoplasm. (C) Ultrastructure of untreated PB-IPC. (D) Penetration of red fluorescent protein (RFP)-labeled mitochondria into PB-IPC. After PB-IPC were treated with RFP-labeled mitochondria for 4 h, confocal microscopy established RFP+ mitochondria infiltrating the cytoplasm (n = 5). Distribution of RFP+ mitochondria inside of a nuclear was represented by an orange arrow. RFP+ mitochondria (red) were colocalized with the Hoechst 33342-labeled nuclear (blue) and the differential interference contrast (DIC) image (left). (E) MitoTracker Red-labeled mitochondria (pink) entered nuclei (blue), and their colocalization was shown by confocal microscopy (n = 5). Isolated mitochondria from platelets were co-cultured with purified nuclei of PB-IPC for 4 h in the presence of serum-free culture medium X-VIVO 15 at 37 °C and 5% CO2. (F) Expression of CXCR4 on the membrane of purified nuclei (n = 4). (G) Mitochondria displaying CXCR4 ligand SDF-1 (n = 4). (H) Blocking experiment with CXCR4 receptor antagonist AMD 3100. The purified PB-IPC’s nuclei were treated with MitoTracker Red-labeled purified mitochondria in the presence or absence of AMD 3100 (30 μM, n = 3). The equal concentration of solvent DMSO served as control. After the treatment for 4 hrs, nuclei were washed twice with PBS and prepared for flow cytometry.
Figure 6
Figure 6
RNA-seq analysis in PB-IPC. Differentially expressed genes shown by the RNA-seq in four PB-IPC preparations treated with mitochondria. Untreated PB-IPC served as controls. (A) The heatmap revealed that forty-six most differentially expressed genes in PB-IPC post the treatment with mitochondria. (B) Thirty-seven up-regulated genes in PB-IPC post the treatment with mitochondria. (C) Nine down-regulated genes in PB-IPC post the treatment with mitochondria.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Nanditha A., Ma R.C., Ramachandran A., Snehalatha C., Chan J.C., Chia K.S., Shaw J.E., Zimmet P.Z. Diabetes in Asia and the Pacific: Implications for the Global Epidemic. Diabetes Care. 2016;39:472–485. doi: 10.2337/dc15-1536. - DOI - PubMed
    1. Xu Y., Wang L., He J., Bi Y., Li M., Wang T., Wang L., Jiang Y., Dai M., Lu J., et al. Prevalence and control of diabetes in Chinese adults. JAMA. 2013;310:948–959. doi: 10.1001/jama.2013.168118. - DOI - PubMed
    1. Lotfy M., Adeghate J., Kalasz H., Singh J., Adeghate E. Chronic Complications of Diabetes Mellitus: A Mini Review. Curr. Diabetes Rev. 2017;13:3–10. - PubMed
    1. DeFronzo R.A., Ferrannini E., Groop L., Henry R.R., Herman W.H., Holst J.J., Hu F.B., Kahn C.R., Raz I., Shulman G.I., et al. Type 2 diabetes mellitus. Nat. Rev. Dis. Primers. 2015;1:15019. doi: 10.1038/nrdp.2015.19. - DOI - PubMed
    1. Wong E., Backholer K., Gearon E., Harding J., Freak-Poli R., Stevenson C., Peeters A. Diabetes and risk of physical disability in adults: A systematic review and meta-analysis. Lancet Diabetes Endocrinol. 2013;1:106–114. doi: 10.1016/S2213-8587(13)70046-9. - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources