Making Platelets in the Culture Dish


Bone marrow-based cells known as megakaryocytes are rather uncommon in bone marrow, but these cells are very important for the health and daily operation of the human body. Megakaryocytes, you see, produce platelets, which are critical to clotting broken blood vessels and wound healing. Generating megakaryocytes in cell culture has proven to be rather difficult, but induced pluripotent stem cells might provide a way to make megakaryocytes in culture.

Megakaryocyte
Megakaryocyte

Platelets_large

The differentiation of induced pluripotent stem cells (iPSCs) into megakaryocytes could potentially create a renewable cell source of platelets for treating patient with “thrombocytopenia,” which is a deficiency of platelets. Zack Wang and his colleagues from Johns Hopkins University in Baltimore, Maryland have developed a protocol to make megakaryocytes in culture from iPSCs. However, more than that, Wang and his co-workers wanted to make patient-specific platelets in culture without using any animal products and with compounds that were approved by the US Food and Drug Association. Such a protocol would demonstrate that using such cells in human patients is feasible and safe.

Wang and his colleagues developed an efficient system that generated megakaryocytes from human iPSCs without the use of animal feeder cells and without animal products (known as xeno-free condition). Several crucial reagents necessary to differentiate iPSCs into megakaryocytes into were replaced with Food and Drug Administration-approved pharmacological reagents that included romiplostim (Nplate, a thrombopoietin analog), oprelvekin (recombinant interleukin-11), and Plasbumin (human albumin). Wang and his group used their method to induce megakaryocytes generation from human iPSCs derived from 23 individuals in two steps: 1) generation of CD34+CD45+ hematopoietic progenitor cells (HPCs) for 14 days; and 2) generation and expansion of CD41+CD42a+ megakaryocytes from HPCs for an additional 5 days. After 19 days, Wang and his group observed abundant CD41+CD42a+ megakaryocytes that also expressed the megakaryocyte-specific cell-surface proteins CD42b and CD61. These cells were also polyploid, which means that they had multiple copies of each chromosome rather than just 2 copies (≥16% of derived cells with DNA contents >4N). Gene expression studies showed that megakaryocytic-related genes were highly expressed in their cultured megakaryocytes.

Characterization of human induced pluripotent stem cell-derived MKs. (A): Representative images of CFU-MK colonies taken from D14 (upper) and D19 (lower) suspension cells. All the colonies containing at least 50 CD41+ cells were considered CFU-MKs. (B): The number of CFU-MK colonies from 1.5 × 105 isolated CD34+ cells on days 14 and 19. The colonies were counted after 12 days of culture from one 35-mm dish. Mean ± SD; n = 3; ∗∗, p < .01. (C): DNA content analysis by flow cytometry on day 19. Left: The whole population stained by propidium iodide. Right: Double staining using CD41-APC and DAPI, gated on CD41+ population. (D): Wright-Giemsa staining of the suspension cells on day 19. Scale bars = 100 μm. Abbreviations: CFU, colony-forming unit; D, day; MKs, megakaryocytes.
Characterization of human induced pluripotent stem cell-derived MKs. (A): Representative images of CFU-MK colonies taken from D14 (upper) and D19 (lower) suspension cells. All the colonies containing at least 50 CD41+ cells were considered CFU-MKs. (B): The number of CFU-MK colonies from 1.5 × 105 isolated CD34+ cells on days 14 and 19. The colonies were counted after 12 days of culture from one 35-mm dish. Mean ± SD; n = 3; ∗∗, p < .01. (C): DNA content analysis by flow cytometry on day 19. Left: The whole population stained by propidium iodide. Right: Double staining using CD41-APC and DAPI, gated on CD41+ population. (D): Wright-Giemsa staining of the suspension cells on day 19. Scale bars = 100 μm. Abbreviations: CFU, colony-forming unit; D, day; MKs, megakaryocytes.

This protocol could be used to further understand the medical conditions that lead to thrombocytopenia. Deeper understanding of these medical conditions will hopefully lead to better treatments of them. Also, Wang’s protocol may lead to the generation of large numbers of platelets in culture that could then be given to patients who need them.

Advertisements

Published by

mburatov

Professor of Biochemistry at Spring Arbor University (SAU) in Spring Arbor, MI. Have been at SAU since 1999. Author of The Stem Cell Epistles. Before that I was a postdoctoral research fellow at the University of Pennsylvania in Philadelphia, PA (1997-1999), and Sussex University, Falmer, UK (1994-1997). I studied Cell and Developmental Biology at UC Irvine (PhD 1994), and Microbiology at UC Davis (MA 1986, BS 1984).