Serum from pregnant donors induces human beta cell proliferation

Figures & data

Figure 1. Maternal human serum increases EndoC-βH1 beta cell proliferation. (a) EndoC-βH1 cells were cultured in 96-well plates in defined serum free conditions, supplemented with 10% fetal bovine serum (FBS), 10% male serum, 10% non-pregnant female serum (NP), or 10% pregnant female human serum (3rd trimester) for seven days. Proliferation was measured by fluorescent click-iT EdU proliferation assay for microplates as measured by the fluorescence signal of Amplex UltraRed reagent, an HRP substrate, where HRP is attached to EdU by click chemistry. Data are combined from two experiments run with the same conditions. (b) The EndoC-βH1 proliferation response to pregnant serum collected from 6 individual donors at the third trimester (3T) was compared to 24–48 hours post-partum (PP). Five of six individual donors showed a significant increase in proliferation relative to defined serum-free culture medium. There was no difference in proliferation between 3T and PP for any donor. For both panels, each dot represents the proliferation response in an individual well of a 96 well plate. Panels A and B are the same data, combined by donor type (A) or split out into individual serum donor (B). Groups were assessed for differences by one-way ANOVA with Tukey’s post hoc pairwise comparisons relative to serum-free. Values are mean ± SEM. *p < .05, **p < .01, ***p < .001.

Table 1. Characteristics of female^a research volunteers.

Donor	Diabetes	Pregnant	Race	Age	GA delivery	BMI delivery	Hyper-tension	GA sample	Glucose	A1c
A − 3T^b	0	1	White	22	35	30.1	0	34	99.9	5.1
A – PP^c	0	0	White	22	35	30.1	0	0	79.5	5.4
B − 3T	0	1	White	23	35	20.7	0	34	79.6	5.0
B – PP	0	0	White	23	35	20.7	0	0	93.9	5.3
C − 3T	0	1	White	29	39	32.7	0	39	91.2	5.5
C – PP	0	0	White	29	39	32.7	0	0	82.6	5.5
D − 3T	0	1	White	31	37	28.9	0	37	80.3	5.5
D – PP	0	0	White	31	37	28.9	0	0	80.5	5.6
E − 3T	0	1	White	23	41	46.9	0	41	77.8	5.2
E – PP	0	0	White	23	41	46.9	0	0	73.7	5.1
F − 3T	0	1	African-American	40	36	35.5	1	36	76.5	5.5
F – PP	0	0	African-American	40	36	35.5	1	0	74.3	5.5
NP^d − 1	0	0	White	26	n/a	n/a	n/a	n/a	n/a	n/a
NP − 2	0	0	White	31	n/a	22.8	n/a	n/a	92.1	5.1
NP − 3	0	0	African American	30	n/a	22.7	n/a	n/a	57.7	4.7
NP − 4	0	0	White	29	n/a	25.2	n/a	n/a	67.7	5
NP − 5	0	0	African American	24	n/a	20.3	n/a	n/a	82.2	5.2

^aA pool of serum from males was purchased from a commercial vendor. The data presented in this table were not available for male serum donors.

^bThird trimester serum collection (3T)

^cPostpartum 24–48 hours serum collection (PP)

^dNon-pregnant female (NP)

Table 2. Human islet donor characteristics.

Donor number	Donor RRID	Age	Sex	BMI	HbA1c	Donor type
1	SAMN13028024	57	female	23.5	4.40%	no diabetes
2	SAMN32641505	16	male	29.5	5.40%	no diabetes
3	SAMN38788294	42	male	20.7	5.5%	no diabetes

Figure 2. Pregnant human serum increases proliferation in primary human beta cells. (a–c) monolayer preparations of primary human islets were cultured with 10% pregnant or 10% non-pregnant serum from five pooled donors for seven days. Proliferation was detected by imaging of EdU incorporation in insulin positive cells. Panel (c) shows increased magnification verifying presence in insulin⁺/EdU⁺ beta cells. (d) Image shows a mitotic figure with condensed chromosomes in an adult human beta cell treated with pregnant serum verifying that beta cells can proliferate under these conditions, albeit at very low frequency. (e) Image quantification of Ins⁺/EdU⁺ cells. Each dot represents EdU positive beta cells for a single image. Islet donor 1: N = 32 and 41 images for non-pregnant and pregnant, respectively, across 9 wells. Islet donor 2: N = 71 and 70 images for non-pregnant and pregnant, respectively, across 15 wells. Islet donor 3 N = 105 and 112 images for non-pregnant and pregnant, respectively, across 8 wells). Groups were assessed for differences by two-tailed t test, p-values are shown on the plots. (f) Image quantification shown as the total cells counted across all images. Each dot represents the fraction of Ins⁺/EdU⁺ cells from all cells imaged in all wells in one islet donor. Groups were assessed for differences by two-tailed t test, p-value is shown on the plot.

Table 3. Human islet monolayer total cell counts following treatment with pooled pregnant or non-pregnant human serum. Data correspond to the plot in figure 2F.

	Islet donor 1		Islet donor 2		Islet donor 3
	Non-pregnant	Pregnant	Non-pregnant	Pregnant	Non-pregnant	Pregnant
total β cells	3547	4430	11250	10218	8028	7723
EdU^+ β cells	8	42	28	95	4	49
fraction Ins^+/EdU^+	0.0023	0.0095	0.0025	0.0093	0.0005	0.0063

Figure 3. Beta cell nuclear doublets in pregnant serum-treated beta cells. Several representative images of beta cell monolayers stained for insulin, EdU incorporation and DAPI for nuclei following culture with 10% non-pregnant (A) or pregnant (B) human serum from pooled donors for one week. The images in panel (B) show evidence of primary human beta cell proliferation by the observation of EdU⁺ cells frequently occurring as doublets (yellow arrows), indicative of two neighboring daughter cells arising from a recent cell division event.