Introduction
Three decades after the ground breaking proposal of using human umbilical cord blood
(UCB) as a source of transplantable hematopoietic stem cells (HSCs),[1 ] over 40,000 transplants have been performed worldwide.[2 ] The hematopoietic reconstitution capacity of UCB resides in the high concentration
of CD34+ cells, enriched for hematopoietic stem and progenitors cells.[3 ]
[4 ]
Since 1993, public UCB banks have been established worldwide. In Italy, UCB is stored,
free of charge, when (1) it is altruistically donated for HSC transplantation and
(2) it is for a family affected with, or at risk of, a disease that is treatable with
transplantation.[5 ]
UCBs that do not meet the requirements for banking based on the international standards
became invaluable sources for stem cell research.[6 ] The study of human hematopoiesis is one of the major applications as this human
model can overcome drawbacks related (1) to the use of animals, which can be poor
predictors of human physiology,[7 ] (2) to the high cost and expertise necessary for generating the embryonic and induced
pluripotent cell lines,[8 ]
[9 ]
[10 ] or (3) to the limited supply of adult HSCs, which usually become available when
discarded after clinical procedures.[11 ]
[12 ]
Different protocols have been established for in vitro megakaryocyte (Mk) differentiation
from CD34+ cells. All entail the usage of various concentrations of recombinant human thrombopoietin
(TPO) in combination with a variety of hematopoietic cytokines but with contrasting
results in terms of Mk and proplatelet phenotypes.[13 ]
[14 ]
[15 ]
[16 ]
We report a retrospective analysis of our 15-year experience in UCB processing, with
a focus on UCB features and experimental procedures that are basic for a reproducible
culture of functional Mks without the need for serum supplementation or coculture
with feeder cells.
Results and Discussion
The UCB bank of the I.R.C.C.S. Policlinico San Matteo Foundation of Pavia, in Italy,
collects 4 UCB units/day. After donor screening and testing for infectious agents,
samples that meet the international standards for banking (currently > 1.6 × 109 total nucleated cells [TNCs] or > 1.2 × 109 TNCs and 2 × 106 CD34+ cells) are stored, while the others are forwarded to research laboratories. Among
these, our laboratory handled a median of 16 unstored samples/month, corresponding
to more than 1,500 UCB units processed in the past 15 years. All the samples were
analyzed within a time-lapse of 0 to 5 days from the date of collection, with most
of them (∼80%) processed within 3 days ([Fig. 1A ]). A retrospective analysis of these samples showed a median volume of 75 mL/unit
and a median cell count of 10 × 103 /μL white blood cells, 3 × 106 /μL red blood cells, and 200 × 103 /μL platelets ([Fig. 1B–E ]). The percentage of CD34+ cells was approximately 0.2% ([Fig. 1B ]). Of these, we separated a median of 0.85 × 106 CD34+ cells/UCB (range: 0.13–5.8 × 106 ), by immunomagnetic sorting procedure. No significant differences were observed in
the number and viability of CD34+ cells isolated from day 0 to 5 ([Fig. 1F, G ]), thus supporting the notion that UCB CD34+ cell survival can last for several days after collection.[1 ]
Fig. 1 General characteristics of umbilical cord blood units. (A ) Percentage of samples processed in the different day intervals. (B ) Median values and range of the volume and total cell count of umbilical cord blood
(UCB) units (WBC, with blood cell; RBC, red blood cell; PLT, platelet). Based on the
concentration of (C ) WBCs, (D ) RBCs, and (E ) PLTs, UCB unit distribute with a Gaussian-like distribution within the range of
analysis. (F ) Box and whisker diagram of the number of CD34+ cells obtain from the UCB samples according to the day interval in which the sample
was processed (p = NS). (G ) Percentage of viable CD34+ cells obtained from the UCB samples according to the day interval in which the sample
was processed. Data are expressed as mean and standard deviation (SD) (p = NS).
Upon harvesting, 1 × 106 CD34+ cells/mL were cultured in a serum-free medium in the presence of 10 ng/mL TPO and
10 ng/mL interleukin (IL)-11, which were renewed every 3 days over 2 weeks of differentiation.
The median number of viable Mks quantified at the end of each culture was 1 × 106 (range: 0.1–7 × 106 ). A significant correlation between the input number of CD34+ cells and the corresponding number of differentiated CD41+ CD42b+ Mks was shown by linear regression analysis (R
2 = 0.85, p < 0.0001; [Fig. 2A ]), regardless of the timing of UCB processing after collection, thus demonstrating
that UCB CD34+ cells maintain full differentiation capability over 5 days after sampling. These
data highlight the efficient rate of success of our culture conditions that support
the differentiation of one mature Mk per starting CD34+ cell, rather than the proliferation of immature progenitors. Comparable results were
obtained after thawing cryopreserved CD34+ cells, consistent with previous knowledge about the efficient recovery of UCB HSCs
after several years of storage.[17 ]
Fig. 2 Megakaryopoiesis from umbilical cord blood hematopoietic stem cells. (A ) CD34+ cells were cultured in a serum-free medium in the presence of interleukin (IL)-11
(10 ng/mL) and thrombopoietin (TPO) (10 ng/mL) for 2 weeks. Linear regression analysis
of the number of CD34+ cells at the input and CD41+ CD42b+ at the output proved to be significant with an R
2 = 0.85. (B ) The diameter of cells was measured randomly throughout the culture, to assess the
rate of maturation. Data are expressed as mean ± standard deviation (SD) (p < 0.05). (C ) Megakaryocyte (Mk) ploidy was quantified at the end of the culture by flow cytometry
by gating CD41+ events within the corresponding parameters of size and complexity to mature Mks.
Data are expressed as mean ± SD. (D ) Panning of cell surface maturity markers on Mks on the 14th day of culture was performed
by flow cytometry. Data are expressed as mean ± SD. (E ) Differentiation was confirmed by fluorescence microscopy (green: CD61; red: CD41;
blue: nuclear stain Hoechst 33258; scale bar: 30 µm). (F ) Representative light microscopy images of proplatelet formation by Mks in liquid
culture (scale bar = 50 µm). (G ) The percentage of proplatelet forming Mks was calculated as the number of cells
displaying long filamentous pseudopods with respect to the total number of round Mks
per analyzed field. Histograms show the percentage of proplatelet formation throughout
the culture. Data are expressed as mean ± SD (*p < 0.01). (H ) Mks at day 13 of culture were plated on fibronectin-coated coverslips. After 30 minutes,
5 hours, or 16 hours of incubation adherent cells were fixed and stained for immunofluorescence
analysis with TRITC-phalloidin (red) and antibody against α-tubulin (green). Nuclei
were counterstained with Hoechst 33258 (blue). Scale bar = 30 µm. (I ) CD34+ cells were cultured in a serum-free medium in the presence of IL-11 (10 ng/mL) and
increasing concentrations of TPO (10–50–100 ng/mL). Analysis of proplatelet structure
was performed after 2 weeks by immunofluorescence staining of the Mk-specific cytoskeleton
component β1-tubulin (green = β1-tubulin; blue = nuclei; scale bar = 25 µm). In all
tested conditions, the representative pictures show similar elongation of proplatelet
shafts with the presence of bulbous tips, at the terminal ends of each branch, resembling
mature platelets. (L ) The analysis of the percentage of proplatelet forming Mks in the different tested
conditions show comparable Mk function. Data are expressed as mean ± SD (p = NS).
During differentiation, a progressive increase in the percentage of cells with high
diameter, ploidy, and expression of lineage-specific markers was observed ([Fig. 2B–E ]). Electron microscopy analysis demonstrated the development of the demarcation membrane
system and the presence of granules throughout the cytoplasm ([Supplementary Fig. S1 ], available in the online version). At the end of the culture > 95% of Mks were viable
([Supplementary Fig. S2 ], available in the online version) and approximately 91 ± 5% of cells expressed late-stage
differentiation markers, such as CD41 and CD42b. Of these, approximately 13 ± 3% Mks
elongated branched proplatelets in liquid culture ([Fig. 2F, G ]). The process of proplatelet formation was spontaneously initiated by Mks and burst
between day 13 and 14 of differentiation, independently of the presence of any cytokines,
including TPO. This was probably due to the regulation of proplatelet formation through
autocrine-paracrine signaling,[18 ]
[19 ] even though the exact mechanisms that drive proplatelet formation are still unknown.[20 ] Platelet-like particles could be found in the culture medium ([Supplementary Fig. S1 ], available in the online version). Additionally, in our three-dimensional silk-based
bone marrow models we demonstrated that UCB-derived Mks release platelets with the
same morphological and functional features of peripheral blood platelets.[21 ]
[22 ]
[23 ]
In vivo Mk function is supported by the interaction with extracellular matrix components.
Among these, fibronectin is known to support proplatelet formation.[13 ]
[24 ] Upon adhesion on fibronectin, we showed that Mks activate different cellular processes:
(1) early passive adhesion; (2) stress fiber formation and microtubule polymerization
with proplatelet-like pseudopod formation; and (3) proplatelet branching ([Fig. 2H ]). Mk cultures with increasing concentrations of TPO, from 10 to 100 ng/mL, did not
prompt further Mk differentiation or proplatelet formation ([Fig. 2I, L ]).
In summary, we developed a protocol to differentiate Mks from UCB CD34+ cells using minimal concentrations of TPO and IL-11. The analysis of 1,500 UCB samples
indicates that our culture protocol is highly reproducible and represents a gold standard
for the study of human megakaryopoiesis. UCB HSCs are cells of fetal/neonatal origin,
and Mks derived from these cells present distinct characteristics such as high proliferation
rate, low ploidy, and mature cytoplasm. For this reason, low-ploidy neonatal Mks are
more mature than adult low-ploidy Mks.[14 ] Despite these differences, our protocol has been designed to promote exclusively
Mk maturation resulting in almost one CD41+ CD42b+ Mk per CD34+ cell. The controlled proliferation in our cultures leads to the production of a uniform
population of CD41+ CD42b+ Mks. The consistency of this protocol makes these Mks a highly reliable tool for
different studies ranging from basic science to disease modeling and drug testing.[12 ]
[13 ]
[25 ]
