Short Article

The postnatal pancreatic microenvironment guides b

cell maturation through BMP4 production
Graphical abstract Authors
Lina Sakhneny, Laura Mueller,
Anat Schonblum, ..., Heather Wilson,
Francesca M. Spagnoli,
Limor Landsman

Correspondence
limorl@tauex.tau.ac.il

In brief
While the postnatal maturation of b cells
is crucial for glucose homeostasis, what
drives it is primarilyunknown. Here,
Sakhneny et al. identified BMP4,
produced by pericytes of the postnatal
and adult pancreas, as a key regulator of
this process. BMP4 induces the
functional maturation of both native and
stem-cell-derived b cells.

Highlights
d BMP4 is produced by pancreatic pericytes from the middle of
the postnatal period

d Postnatal BMP4 is required for insulin biosynthesis and

glucose regulation

d The expression of core b cell genes is regulated by BMP4

downstream signaling

d BMP4 promotes the functional maturation of human stem-

cell-derived b cells

Sakhneny et al., 2021, Developmental Cell 56, 1–9

October 11, 2021 ª 2021 Elsevier Inc.
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Short Article
The postnatal pancreatic microenvironment
guides b cell maturation through BMP4 production
Lina Sakhneny,1 Laura Mueller,2 Anat Schonblum,1 Sivan Azaria,1 Guzel Burganova,1 Alona Epshtein,1 Abigail Isaacson,2
Heather Wilson,2 Francesca M. Spagnoli,2 and Limor Landsman1,3,*
1Department of Cell and Development Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
2Centre for Stem Cell and Regenerative Medicine, King’s College London, Great Maze Pond, London SE1 9RT, UK
3Lead contact

*Correspondence: limorl@tauex.tau.ac.il
https://doi.org/10.1016/j.devcel.2021.08.014

SUMMARY

Glucose homeostasis depends on regulated insulin secretion from pancreatic b cells, which acquire their
mature phenotype postnatally. The functional maturation of b cells is regulated by a combination of cell-
autonomous and exogenous factors; the identity of the latter is mostly unknown. Here, we identify BMP4
as a critical component through which the pancreatic microenvironment regulates b cell function. By
combining transgenic mouse models and human iPSCs, we show that BMP4 promotes the expression of
core b cell genes and is required for proper insulin production and secretion. We identified pericytes as
the primary pancreatic source of BMP4, which start producing this ligand midway through the postnatal
period, at the age b cells mature. Overall, our findings show that the islet niche directly promotes b cell
functional maturation through the timely production of BMP4. Our study highlights the need to recapitulate
the physiological postnatal islet niche for generating fully functional stem-cell-derived b cells for cell
replacement therapy for diabetes.

INTRODUCTION Activation of the BMP4/BMPR1A downstream signaling has

Mature b cells, which populate the adult pancreas, are capable of
accurate insulin secretion in response to elevated glucose levels.
b cells differentiate during embryogenesis but acquire their mature,
functional phenotype only after birth. b cell maturation begins
midway through the postnatal period and is mostly completed by
the end of this period (Blum et al., 2012; Stolovich-Rain et al.,
2015). This process is defined by increased insulin production
and coordinated glucose-stimulated insulin secretion (GSIS) (Bon-
ner-Weir and Aguayo-Mazzucato, 2016; Gregg et al., 2012). Once
established, b cell maturity needs to be continuously maintained
(Landsman et al., 2011; Talchai et al., 2012). While several cell-
autonomous factors required for b cell functional maturation were
identified (Arda et al., 2016; Bevacqua et al., 2021; Blum et al.,
2012), cues provided by the islet niche driving this process are
unknown.
Restoring glucose regulation in individuals with diabetes requires
replenishing functional b cell mass (Sneddon et al., 2018).
Protocols to generate stem-cell-derived cells aim to recapitulate
b cell development by relying on the molecular mechanisms that
naturally govern this process. However, stem-cell-derived cells
mostly resemble immature rather than mature b cells (Nair et al.,
2020; Sneddon et al., 2018). Thus, employing factors that govern
the physiological, postnatal b cell maturation can promote this
process in stem-cell-derived cells, enhancing their resemblance
to endogenous pancreatic b cells and improving their insulin
secretion.
been implicated in b cell function (Goulley et al., 2007). However,
studies aiming to determine the influence of this pathway on b
cells have produced conflicting results, implicating either sup-
portive or destructive roles. Genetic deletion of BMPR1A caused
b cell dysfunction (Goulley et al., 2007; Jiang et al., 2015),
whereas its pharmacological activation in stressed b cells pro-
moted their dedifferentiation (Triñanes et al., 2019). Prolonged
exposure of cultured b cells to BMP4 curbed their insulin secre-
tion capabilities (Bruun et al., 2014; Christensen et al., 2015),
while systemic administration of this ligand improved insulin
secretion in vivo (Goulley et al., 2007; Hoffmann et al., 2020;
Sakhneny et al., 2018). These discrepancies imply that the b
cell’s response to BMP4 is context-dependent, as recently sug-
gested for its family of ligands (Antebi et al., 2017). However, the
pancreatic source of BMP4 and the context in which it is naturally
produced are yet to be characterized.
Islets are highly vascularized by a capillary network composed
of endothelial cells and pericytes. Recent studies by us and
others ascribed multiple roles to pericytes in glucose homeosta-
sis, indicating they support b cell function and gene expression
independently of blood-flow regulation (Almaça et al., 2020; Bur-
ganova et al., 2021; Epshtein et al., 2017a; Houtz et al., 2016;
Sakhneny et al., 2018, 2021; Sasson et al., 2016). The molecular
basis of pericytes’ ability to support b cell function has only
recently begun to be unveiled. For instance, pericyte-mediated
b cell function depends on the diabetes-associated transcription
factor TCF7L2, which regulates the expression of an array of

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Figure 1. Pancreatic pericytes produce BMP4 in an age-dependent manner to activate downstream signaling in endocrine cells
(A) Bar diagram (mean ± SD) shows qPCR analysis of bulk pancreatic tissues, isolated islets, pancreatic endothelial cells (ECs), and pancreatic pericytes of adult
mice, normalized to Gapdh. N = 3.

(legend continued on next page)

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Short Article

pericytic-secreted factors; among them is BMP4 (Sakhneny
et al., 2018). However, whether and how the islet vasculature
drives b cells functional maturation is yet to be reported.
Here, we set to determine if BMP4 is required for b cells func-
tional maturity. First, we characterized the pancreatic source of
BMP4 and found pericytes to be the primary cell population to
produce it. Importantly, pericytes produce BMP4 in an age-
dependent manner from the late postnatal period, coinciding
with b cell maturation. Next, we employed transgenic mouse
systems and human iPSC-derived cells to determine the role
of BMP4. Mice lacking pericytic BMP4 were glucose intolerant
due to diminished insulin biosynthesis, which was associated
with impaired expression of core b cell genes. To assess whether
BMP4 can actively drive the functional maturation of b cells,
differentiated human iPSCs-derived b cells were exposed to
this factor, which improved their expression of core b cell genes
and GSIS capability. Thus, BMP4, naturally produced postna-
tally by pancreatic pericytes, promotes the mature b cell
phenotype in both humans and mice.
RESULTS
Pericytes are a primary pancreatic source of BMP4
The source of BMP4 in the adult pancreas is unclear. BMP4 has
been proposed to influence b cells in an autocrine manner;
however, recent transcriptome studies unveiled its low expres-
sion levels in these cells (Bruun et al., 2014; Goulley et al., 2007;
Jiang et al., 2015; Segerstolpe et al., 2016). By contrast, Bmp4
was shown to be expressed by pancreatic pericytes (Sakhneny
et al., 2018), and it is the predominant BMP ligand produced by
this cell population (Figure S1). To determine the contribution of
pericytes to BMP4 production in the pancreas, we compared its
expression in various cell populations. Bmp4 expression levels
in pericytes were two orders of magnitude higher than in the other
tested pancreatic cell types (Figure 1A). Similarly, immunofluores-
cent analysis detected BMP4 in pericytes but not in other islet cell
populations, including the closely related vascular smooth muscle
cells (Figures 1B and 1C). Thus, pericytes produce BMP4 and
constitute its primary source in the pancreas.
Pancreatic pericytes express BMP4 in an age-
dependent manner
Next, we determined the age(s) at which pancreatic pericytes
produce BMP4. Minute amounts of Bmp4 transcript were de-
tected in cells from embryonic (embryonic day [e] 17.5) and
newborn (postnatal day [p] 0) pancreata, while its levels rose
2.5-fold at the middle of the neonatal period (p10) and 12-fold
by its end (p21) (Figure 1D). Bmp4 transcript levels remained un-
changed from the end of the neonatal period to adulthood (Fig-
ure 1D). In summary, pericytes produce BMP4 in an age-depen-
dent manner, gradually increasing from the middle of the
neonatal period. Interestingly, the timing of pericytic BMP4
expression highly resembles that of b cell maturation (Blum
et al., 2012).
Activation of the BMP signaling pathway in islets
depends on pericytic BMP4
In the adult pancreas, activation of the BMP4 pathway was
shown to be restricted to a and b cells (Goulley et al., 2007).
Accordingly, islets express the BMP4 receptor Bmpr1a and dis-
played activation of the pathway, as indicated by Smad1/5/9
phosphorylation (Figures 1E and 1F). To determine the role of
pancreatic pericytic BMP4, we deleted this factor in these cells
by crossing Bmp4flox mice with the Nkx3-2-Cre line (Harari
et al., 2019; Sasson et al., 2016; Verzi et al., 2009) to generate
DBmp4peri (Nkx3-2-Cre;Bmp4flox/flox) mice (Figure 1G).
DBmp4peri islets displayed lower levels of phosphorylated
Smad1/5/9 than control (Cre-negative Bmp4flox/flox) accompa-
nied by a reduced expression of the pathway target gene Id2
(Figures 1F and 1H). Thus, our analysis indicates that activation
of the BMP downstream signaling in pancreatic endocrine cells
is regulated by pericytic BMP4 production.
Reduced blood insulin levels in mice lacking
pericytic BMP4
To directly determine the role of pericytic BMP4 in glucose
regulation, we analyzed females and males DBmp4peri and
non-transgenic control mice (Figures 2A–2E). Transgenic
male mice had significantly higher fed and fasted glucose levels
than sex-matched controls and displayed impaired glucose
tolerance but intact insulin sensitivity (Figures 2C–2E). Interest-
ingly, the glucose response of DBmp4peri female mice was un-
affected (Figures 2A and 2B). Female and male mice expressed
comparable levels of BMP4 and downstream genes (Figure S1),
suggesting their different phenotypes resulted from the docu-
mented sex-dependent variances in glucose metabolism (Ma-
cotela et al., 2009). Correlating with their hyperglycemia,
DBmp4peri male mice had significantly lower fed and fasted
serum insulin levels than controls and displayed a blunted
GSIS (Figures 2F and 2G). In contrast, serum glucagon levels
were comparable in DBmp4peri and control male mice (Fig-
ure 2H). Together, our analyses point to impaired glucose
regulation in the absence of pericytic BMP4 due to lower blood
insulin levels.

(B) Immunofluorescence analysis of mouse pancreatic tissue sections for BMP4 (green). The pericytic marker NG2 (red), insulin (white), and DAPI (blue). Left
panel, Scale bar size: 50 mm. The right panel shows higher magnification of the area framed in the white box in the left panel. Scale bar size: 10 mm.
(C) Immunofluorescence analysis of mouse pancreatic tissue sections for BMP4 (green), aSMA (red), PECAM1 (white), and DAPI (blue). Scale bar size, 50 mm.
(D) Bar diagram (mean ±SD) showing qPCR analysis of Bmp4 expression in pancreatic pericytes at embryonic day (e) 17.5, postnatal day (p) 0 (average was set to
‘‘1’’), p10, p21, and adulthood. N = 4–5.
(E) Bar diagram (mean ±SD) showing pancreatic Bmpr1a expression in bulk pancreatic tissues and isolated islets of adult mice, normalized to GAPDH (N = 3–4).
(F) Western blot analysis for phospho-Smad1/5/9 (upper panels) and Smad1 (lower panels) of islets protein extract from DBmp4Peri and non-transgenic (‘‘non tg’’)
adult male mice. A representative of two independent experiments.
(G and H) Bar diagram (mean ±SD) showing Bmp4 and Id2 expression in pericytes (G) and islets (H), respectively, of DBmp4Peri mice (blue) and non-transgenic
(‘‘non tg’’; gray; the average was set to ‘‘1’’) adult male mice. N = 3–5.
*p < 0.05, **p < 0.01, ***p < 0.005; NS, not significant (Student’s t test). Each dot represents a single sample. See also Figure S1.

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Figure 2. Blunted insulin-dependent glucose regulation in mice lacking pericytic BMP4

13-week-old DBmp4Peri female (magenta; A and B) and male (blue; C–H) mice, which lack Bmp4 expression in their pancreatic pericytes, and sex-matched
non-transgenic littermates (Cre-negative; ‘‘non tg’’; gray) were analyzed.
(A and C) Bar diagrams (mean ±SD) show blood glucose levels after an overnight fast or upon ad-lib feeding. N = 9–11.
(B and D) Intraperitoneal glucose tolerance test (IPGTT). Shown are mean (± SEM) blood glucose levels. N = 11.
(E) Intraperitoneal insulin tolerance test (ITT). Shown are mean (± SEM) blood glucose levels. N = 7–9.
(F) Bar diagram (mean ±SD) shows serum insulin levels after an overnight fast or upon ad-lib feeding. N = 6–9.
(G) In vivo glucose-stimulated insulin secretion (GSIS). Shown are mean ± SEM. N = 11.
(H) Bar diagrams (mean ±SEM) show serum glucagon levels after an overnight fast. N = 6.
*p < 0.05, **p < 0.01, ***p < 0.005, as compared with non-transgenic mice (Student’s t test). Each dot represents a single mouse.

Pancreatic insulin production is regulated by
pericytic BMP4
Next, we tested the underlying causes of impaired blood insulin
levels of DBmp4peri male mice. The functionality and density of
the islet vasculature, as well as islet pericytic coverage, were un-
affected in DBmp4peri mice (Figure S2). When exposed to high
glucose levels, islets isolated from DBmp4peri mice secreted
less insulin (Figure 3A), which indicated that the observed
impaired GSIS is blood-flow independent. Insulin contents
were lower in adult DBmp4peri islets and pancreata (Figures 3B
and 3C). Importantly, when normalized to its content, the amount
of insulin secreted in response to glucose challenge was compa-
rable in DBmp4peri and control islets (Figure 3D). However, such
normalization revealed that transgenic islets secrete more insulin
in the presence of low glucose levels, which is a characteristic of
immature b cells (Figure 3D).
Islet morphology, b- cell mass, and a/b cells ratio were compa-
rable in DBmp4peri and control mice (Figure S3). Nevertheless,
DBmp4peri islets had a lower proinsulin content (Figure 3E).
Interestingly, the insulin-to-proinsulin ratio was comparable in
transgenic and control islets (Figure 3F). We further detected
lower transcript levels of Ins1, Ins2, and Psck1 (encoding insulin
and proprotein convertases 1/3, respectively) in BMP4-deficient
islets (Figure 3G). The expression of genes encoding glucagon,
somatostatin, and components of the GSIS machinery was unaf-
fected in DBmp4peri islets (Figures 3H and S3), further pointing to
impaired insulin production as the primary cause of mice glucose
intolerance.
Insulin production is regulated by an array of transcription fac-
tors expressed by mature b cells (Bramswig and Kaestner,
2014). Transgenic islets had lower transcript levels of MafA,
Pdx1, NeuroD1, and Nkx6-1 (Figure 3I); all are directly involved
in insulin transcription. In contrast, the expression of Ucn3,
expressed by mature b cells, was unaffected (Figure 3I). Pharma-
cological inhibition of BMPR1A activity in cultured islets similarly
hampered the expression of core b cell genes (Figure 3J), point-
ing to the continuous requirement of the BMP4/BMPR1A
pathway in these cells.

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Figure 3. Impaired insulin production and core b cell gene expression in DBmp4Peri islets
Islets and pancreatic tissues of DBmp4Peri transgenic (blue; A–I) and non-transgenic (‘‘non-tg’’; gray) male mice were analyzed.
(A) Bar diagram (mean ± SD) shows GSIS of adult isolated islets. N = 4–5.
(B) Bar diagram (mean ±SD) shows insulin content of adult isolated islets. N = 5.
(C) Bar diagram (mean ±SD) shows pancreatic insulin content in adult (13-weeks-old) and newborn (p0) mice, normalized to protein content. N = 4–5.
(D) Bar diagrams (mean ±SD) show secreted insulin (as shown in A0 ) normalized to its content (as shown in B0 ).
(E) Bar diagrams (mean ± SD) show islet proinsulin content. N = 6–7
(F) Bar diagrams (mean ±SD) show islet insulin-to-proinsulin ratio. N = 5.
(G–I) Bar diagrams (mean ±SD) show core b cell gene expression. Average levels in control islets were set to ‘‘1.’’ N = 5–8.
(J) Cultured non-transgenic islets were either treated with BMPR1A inhibitor (purple) or left untreated (gray; the average was set to ‘‘1’’). Bar diagrams (mean ± SD)
show expression levels of indicated genes. n = 5.
*p < 0.05, **p < 0.01, ***p < 0.005; NS, not significant, as compared with non-transgenic control mice or untreated cultured islets (Student’s t test). See also Figures
S2 and S3.

Thus, in the absence of pericytic BMP4, reduced expression of
mature b cell transcription factors and impaired insulin produc-
tion leads to the irregular secretion of this hormone.
Pericytic BMP4 is required postnatally
Our analysis indicated that pancreatic pericytes express BMP4
postnatally (Figure 1D); thus, the observed phenotype is unlikely
to reflect developmental defects. Indeed, DBmp4peri newborns
(p0) displayed comparable b cell mass and pancreatic insulin
content to control (Figures 3C and S3). In agreement with their
normal b cell mass in adulthood, the b cell proliferation rate
was unaffected in DBmp4peri pups (p14; Figure S3). To directly
test the postnatal requirement of pericytic BMP4, we
crossed the Bmp4flox mouse line with an inducible pericytic
Cre mouse line, Myh11-CreERT, to generate iDBmp4peri
(Myh11-CreERT;Bmp4flox/flox) male mice, which were treated
with tamoxifen at p21 or left untreated. Glucose and insulin toler-
ance tests indicated that tamoxifen-treated iDBmp4peri mice

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Figure 4. BMP4 treatment promoted the functional maturation of iPSC-derived b-like cells
(A) A schematic diagram of the stepwise differentiation process of iPSCs toward b-like cells. The period of recombinant human BMP4 (rBMP4) treatment (d17-19)
is marked with a green box.
(B–E) iPSCs at days 0 (average was set to ‘‘1’’) and 21 of the differentiation process, the latter either untreated (gray) or treated with rBMP4 (green). Bar diagrams
(mean ± SD) show gene expression analysis. n = 3. Shown is a representative of three independent experiments.
(F) Scatter plot shows the portion of insulin+ cells derived from iPSCs at d21 of the differentiation process, either untreated (gray) or rBMP4 treated (green).
(G) Bar diagram shows the ratio between insulin secretion in response to high (20 mM) and low (2 mM) glucose levels from b-like cells, either untreated (gray) or
treated with rBMP4 (green). n = 3. Shown is a representative of two independent experiments.
*p < 0.05, **p < 0.01, ***p < 0.005; NS, not significant (Student’s t test). See also Figure S4.

were glucose intolerant but insulin sensitive (Figure S3). Further,
Ins1 expression was significantly lower in islets of these mice
(Figure S3). Thus, our results suggest that pericytic BMP4 is
required after birth, whereas embryonic b cell development is
unaffected.
BMP4 promotes the maturation of iPSC-derived b cells
To directly study if BMP4 can drive b cell maturation, we
extended our study to human cells. To this end, human iPSCs
were differentiated toward a b cell fate according to previously
established protocols (Helker et al., 2019; Russ et al., 2015) (Fig-
ure 4A), and derived cells were analyzed at the onset (d0),
midway (d14; the endocrine precursor stage), and end (d21;
the b-like cell stage) of the protocol. At the end of the differenti-
ation process, iPSCs-derived cells expressed insulin, transcrip-
tion factors, and components of the GSIS machinery, pointing
to their differentiation into b-like cells (Figures 4B–4E). In contrast
to endocrine precursors, human iPSC-derived b-like cells
expressed low levels of BMP4 and its direct target gene ID2 (Fig-
ure S4). However, BMPR1A transcript levels were upregulated
as iPSCs differentiated to endocrine precursors and b-like cells
(Figure S4).
To recapitulate the physiological stage at which b cells are
exposed to pericytic BMP4, iPSCs-derived cells were treated
with recombinant BMP4 (rBMP4) during the final step of the dif-
ferentiation protocol, after the acquisition of a b cell identity (i.e.,
from day 17 to 19; Figure 4A) or left untreated. rBMP4 treatment
did not alter the number of b-like cells (identified as insulin+ or

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PDX1+NKX6-1+ cells; Figures 4F and S4, respectively), indi-
cating that the differentiation process was indeed unaffected.
However, rBMP4 enhanced the expression of an array of genes
required for b cell function, including INS and components of the
GSIS machinery, as well as core b cell transcription factors (Fig-
ures 4B–4E). Finally, rBMP4-treated b-like cells displayed
improved GSIS capabilities compared with those of untreated
controls (Figure 4G).
To conclude, our results indicated that exogenous BMP4
induces the functional maturation of iPSC-derived b-like cells
and, consequently, improves their response to glucose. This
set of analyses further suggests that the requirement of BMP4
for b cell maturation is conserved between mice and humans.
DISCUSSION
Here, we show that pericytes regulate b cell functional matura-
tion through the production of BMP4. Pericytes produce BMP4
postnatally and represent the primary source of this ligand in
the mouse pancreas. Loss of pericytic BMP4 in transgenic
mice hampered GSIS in vivo and ex vivo due to impaired insulin
production and secretion. b cell dysfunction was associated with
lower expression levels of transcription factors required for these
cells’ functional maturity. Importantly, we found that BMP4
drives b cell maturation and improved insulin secretion also in
the human iPSC-derived b cell model system. To conclude,
our findings indicate that BMP4, naturally produced by
pancreatic pericytes, induces the functional maturation of both
human and mouse b cells.
We suggest that pericytic BMP4 acts directly on b cells to
regulate their gene expression and function. Indeed, proper acti-
vation of the downstream signaling in islets required pericytic
BMP4 production (Figure 1). Further, rVISTA analysis (Loots
and Ovcharenko, 2004) predicts SMAD binding sites in the pro-
moter regions of human NEUROD1 and MAFA, suggesting they
are direct target genes of the BMP4/BMPR1A pathway.
Correspondingly, our analysis shows that BMP4 regulates the
expression of these transcription factors (Figures 3 and 4),
both directly regulating insulin gene expression (Bramswig and
Kaestner, 2014). Thus, it is probable that BMP4 promotes insulin
production by directly regulating the expression of b cell tran-
scription factors. However, as BMP signaling converges to other
pathways associated with insulin secretion (Jiang et al., 2015),
an indirect effect of pericytic BMP4 on b cells cannot be
ruled out.
The influence of BMP4 downstream signaling on b cells
depends on the timing of activation when differentiation and
maturation processes are differentially affected. Early in devel-
opment, the BMP pathway inhibits the acquisition of pancreatic
and endocrine fates (Hua et al., 2006; Russ et al., 2015; Spagnoli
and Brivanlou, 2008), whereas adult b cell function relies on
proper activation of this pathway (Goulley et al., 2007; Henley
et al., 2012; Jiang et al., 2015; Sakhneny et al., 2018; Scott
et al., 2009). Interestingly, BMP4 inhibited the proliferation of
cultured neonatal immature b cells (Christensen et al., 2015),
further implying the influence of timing of exposure to this ligand.
Our data indicate that the production of BMP4 by pericytes fol-
lows a temporal pattern, which coincides with the b cell matura-
tion process (Blum et al., 2012). This pattern may reflect a distinct
ll
requirement during the various stages of b cell life. Indeed, our
data demonstrate that pericytic BMP4 is required for adult, but
not neonatal, insulin production and is dispensable for b cell
proliferation and mass (Figures 3 and S3). Accordingly, exposure
to BMP4 after the differentiation of iPSC-derived b cells pro-
motes the maturation of these cells without affecting their differ-
entiation (Figure 4). Thus, we suggest a temporal-specific role for
BMP4 after b cells have differentiated and proliferated.
Protocols to generate stem-cell-derived b cells in vitro aim to
mimic pancreatic b cell development (Sneddon et al., 2018).
Here, we present a proof-of-concept study showing that recapit-
ulating the postnatal b cell niche by providing cues naturally pro-
duced by cells of the islet microenvironment promotes these
cells’ functional maturation. Furthermore, our data implicate
conservation between the requirement of mouse and human b
cells. Thus, a better characterization of the cues provided by
the islet niche, including pericytes, is crucial for the generation
of fully mature stem-cell-derived b cells for future cell replace-
ment therapy for diabetes.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d RESOURCE AVAILABILITY
B Lead contact
B Materials availability
B Data and code availability
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Mice
B Human iPSCs
d METHOD DETAILS
B Mouse treatments
B Cell and islet isolation
B In vitro glucose-stimulated insulin secretion
B Measurement of hormones secretion and content
B Western blot analysis
B Gene expression
B Islet BMP signaling inhibition
B Immunofluorescence
B Morphometric analysis
B Differentiation of human iPSCs into pancreatic b-
like cells
d QUANTIFICATION AND STATISTICAL ANALYSIS
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.
devcel.2021.08.014.
ACKNOWLEDGMENTS
We thank Jana Omar (Tel Aviv University) for her technical assistance. This
study was supported by the Israel Science Foundation (ISF; grant agreement
no. 1605/18, to L.L), EFSD (grant agreement no. 1117775, to F.S.), JDRF Inno-
vative grant (grant agreement no. 1-INO-2018-634-A-N, to F.S.), and the Zvi
Yanai Scholarship for Israeli Arab, Druze, and Circassian students from the
State of Israel Ministry of Science and Technology (to L.S.). This work was car-
ried out in partial fulfillment of the requirements for a Ph.D. degree for L.S. from
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the Sackler Faculty of Medicine at Tel Aviv University. Graphical abstract was
created with BioRender.com.
AUTHOR CONTRIBUTIONS
Conceptualization, L.L.; investigation, L.S., L.M., A.S., S.A, A.E, A.I., and H.W;
formal analysis, L.S., L.M., A.S., S.A., G.B., and F.M.S.; writing – original Draft,
L.S., and L.L; writing – review & editing, A.S., S.A., A.E., and F.M.S.; funding
acquisition, L.S., F.M.S., and L.L.; supervision, F.M.S. and L.L.
DECLARATION OF INTERESTS
The authors declare no competing interests.
INCLUSION AND DIVERSITY
We worked to ensure sex balance in the selection of non-human subjects. One
or more of the authors of this paper self-identifies as an underrepresented
ethnic minority in science. One or more of the authors of this paper received
support from a program designed to increase minority representation in
science.
Received: February 7, 2021
Revised: July 11, 2021
Accepted: August 16, 2021
Published: September 8, 2021
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Developmental Cell (2021), https://doi.org/10.1016/j.devcel.2021.08.014

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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Rabbit anti- aSMA Abcam Cat #ab5694; RRID: AB_2223021
Mouse anti-Bmp4 Millipore Cat # MAB1049; RRID: AB_2258988
Rabbit anti-Glucagon Millipore Cat # AB932; RRID: AB_2107329
Rabbit anti- Glucagon Immunostar Cat #20076; RRID: AB_572241
Guinea pig anti-insulin Dako Cat: #A0564; RRID: AB_10013624
Guinea pig anti-insulin Dako Dako, #IR00261-2; RRID: AB_2800361
Guinea pig anti-INSULIN Invitrogen Cat #PA1-26938; RRID: AB_794668
Mouse anti-Ki67 BD Bioscience Cat # 550609; RRID: AB_393778
Rabbit anti-NG2 Millipore, AB5320 Cat# AB5320; RRID:AB_11213678
Mouse anti-NKX6-1 Hybridoma Bank Cat #F55A10; RRID: AB_532378
Guinea pig anti-PDX1 Abcam Cat #ab47308; RRID: AB_777178
Rat anti-PECAM1 BD Bioscience Cat #553370; RRID: AB_396660
Rat anti-PECAM1, PE-conjugated BioLegend Cat #102407; RRID: AB_312902
Rabbit anti-pSmad1/5/9 Cell Signaling Technology Cat #13820T; RRID: AB_2493181
Rabbit anti-Smad1 Cell Signaling Technology Cat #6944T; RRID: AB_10858882
Donkey Alexa Fluor 488-labeled anti- Dianova Cat #706-545-148; RRID: AB_2340472
Guinea Pig IgG
Donkey Alexa Fluor 647-labeled anti- Dianova Cat #706-605-148; RRID: AB_2340476
Guinea Pig IgG
Goat Alexa Fluor 555-labeled anti- Guinea Invitrogen Cat #A21435; RRID: AB_2535856
pig IgG
Goat Alexa Fluor 633-labeled anti- Guinea Invitrogen Cat # A21105; RRID: AB_2535757
pig IgG
Goat Alexa Fluor 488-labeled Invitrogen Cat #A11029; RRID: AB_2534088
anti-mouse IgG
Goat Alexa Fluor 488-labeled Invitrogen Cat # A11034; AB_2576217
anti-rabbit IgG
Goat Alexa Fluor 555-labeled Invitrogen Cat #A21429; RRID: AB_2535850
anti-rabbit IgG
Donkey Alexa Fluor 647-labeled Invitrogen Cat #A31573; RRID: AB_2536183
anti-Rabbit IgG
Goat Alexa Fluor 488-labeled anti-rat IgG Invitrogen Cat #A11006; RRID: AB_2534074
Goat Alexa Fluor 555-labeled anti-rat IgG Invitrogen Cat #A21434; RRID: AB_2535855
Goat Alexa Fluor 633-labeled anti-Rat IgG Invitrogen Cat #A21094; RRID: AB_2535749
Chemicals, Peptides, and Recombinant Proteins
10% Mini-PROTEAN TGX Precast Bio-Rad Cat #4561033
Protein Gels
a-Amyloid Precursor Protein Modulator - Sigma-Aldrich Cat #565740-1MG
Calbiochem
ALK5 Inhibitor II Enzo Life Sciences Cat #ALX-270-445-M001
CAS-Block Histochemical Reagent Invitrogen Cat # 008120
Collagenase P Roche Cat #11213865001
CTS B-27 Supplement, XenoFree (100X) ThermoFisher Scientific Cat # A1486701
D-(+)-Glucose Sigma-Aldrich Cat #G8270
Dextrose Sigma-Aldrich Cat #D9434
Deoxyribonuclease I from bovine Sigma-Aldrich Cat #D5025
pancreas (DNase)
(Continued on next page)

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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
HCS CellMask Deep Red Stain Invitrogen Cat #H32721
Histopaque-1119 Sigma-Aldrich Cat #11191
Hoechst 33342 Invitrogen Cat #H1399
Humulin (human insulin) Lilly Cat #0210
Insulin-Transferrin-Selenium ThermoFisher Scientific (Life Technologies) Cat #41400-045
Supplement (100X)
LDN-193189 BMPR1A inhibitor Tebu-Bio Cat #24804-0074
LDN-193189 BMPR1A inhibitor Sigma-Aldrich Cat #SML0559-5MG
Lycopersicon Esculentum (Tomato) Lectin, Vector Laboratories Cat # FL-1171
Fluorescein
Recombinant Human/Mouse/Rat Activin A R&D Systems Cat #338-AC-050/CF
Protein, carrier-free
Recombinant Human BMP4 R&D Systems Cat #314-BP-010
Recombinant Human EGF R&D Systems Cat #236-EG-200
Recombinant Human Heregulin b-1 Peprotech Cat #100-03-10
Recombinant Human KGF/FGF-7 Protein R&D Systems Cat #251-KG-050
Recombinant Human Wnt3a Protein, R&D Systems Cat #5036-WN-010/CF
carrier-free
(ROCK) inhibitor Y-27632 Sigma-Aldrich Cat #SCM075
Signalfire ECL Reagent Cell Signaling Technology Cat # 6883P3
Tamoxifen Sigma-Aldrich Cat #T5648
TGFb RI Kinase Inhibitor IV Millipore Cat #616454-2MG
TTNPB, stable retinoid analog Sigma-Aldrich Cat # T3757-10MG
Critical Commercial Assays
Fast SYBR Green Master Mix Applied Biosystems Cat # 4385612
Fast TaqMan Advanced Master Mix Applied Biosystems Cat # 4444557
High-Capacity cDNA Reverse Applied Biosystems Cat # 4374966
Transcription Kit
Human Insulin ELISA Mercodia Cat #10-1132-01
Mouse glucagon ELISA Alpco Cat #48-GLUHU-E01
Mouse proinsulin ELISA Alpco Cat #80-PINMS-E01
Mouse ultrasensitive Insulin ELISA Alpco Cat #80-INSMSU-E01
Pierce BCA Protein Assay Kit ThermoFisher Scientific Cat #23227
PureLink RNA Micro Scale Kit Invitrogen Cat #12183016
PureLink RNA Mini Kit Invitrogen Cat # 12183018A
STELLUX Chemo rodent insulin Alpco Cat #80-INSMR-CH01
Deposited data
RNAseq data (Sakhneny et al., 2018) https://www.ebi.ac.uk/arrayexpress/
experiments/E-MTAB-5325/)
Experimental models: Cell lines
BIHi043-A (XM001) (Wang et al., 2018) N/A
Experimental models: Organisms/strains
Mouse: Nkx3.2-Cre; (Verzi et al., 2009) Warren Zimmer
Nkx3–2tm1(cre)Wez (Texas A&M University, TX)
Mouse: R26R-EYFP; The Jackson Laboratory Cat #JAX:006148;
Gt(ROSA)26Sortm1(EYFP)Cos RRID:IMSR_JAX:006148
Mouse: Bmp4flox; The Jackson Laboratory Cat #JAX:016878;
B6;129S4-Bmp4tm1Jfm/J RRID:IMSR_JAX:016878
Mouse: Myh11-CreERT2; The Jackson Laboratory Cat #JAX:019079;
B6.FVB-Tg(Myh11-cre/ERT2)1Soff/J RRID:IMSR_JAX:019079
(Continued on next page)

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Developmental Cell (2021), https://doi.org/10.1016/j.devcel.2021.08.014

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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Mouse: wild-type Envigo Ltd Order code 043
C57BL/6JRccHsd
Oligonucleotides
Human primers for RT-qPCR This paper See Table S1
Mouse primers for RT-qPCR This paper See Table S2
Software and algorithms
GraphPad Prism 8 and 9 GraphPad RRID:SCR_002798
ImageJ 1.53 ImageJ RRID; SCR_003070

RESOURCE AVAILABILITY

Lead contact
Further information and requests for resources and reagents should be directed to and will befulfilled by the Lead Contact, Limor
Landsman (limorl@tauex.tau.ac.il).

Materials availability
This study did not generate new unique reagents.

Data and code availability

This study did not generate datasets.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Mice
All experimental protocols were approved by the Tel Aviv University Institutional Animal Care and Use Committee (IACUC). The study
was carried out in compliance with the ARRIVE guidelines. Mice were housed in a specific pathogen-free, temperature-controlled
facility with a 12-h light/dark cycle in individually ventilated cages. Animals were provided food (standard rodent chow diet) and
water ad libitum. The data presented in this study were derived from male or female mice, sex-matched littermates at various
ages (as indicated in the figure legends), assigned to experimental groups according to their genotype. For tamoxifen treatment
experiments, data were derived from 13-weeks-old male mice randomly assigned to experimental groups.

Human iPSCs
Human iPSC line [BIHi043-A (XM001)] was employed (sex: female) (Wang et al., 2018), which is registered in the European Human
Pluripotent Stem Cell Registry (hPSCreg) (https://hpscreg.eu/cell-line/HMGUi001-A) and authenticated by karyotyping. Human iPSC
lines were maintained on Geltrex-coated (Invitrogen) plates in homemade E8 media under hypoxic conditions. The medium was
changed daily, and cells were passaged every 3 days as cell clumps or single cells using 0.5 mM EDTA (Invitrogen) or Accutase
(Invitrogen), respectively. The medium was supplemented with 10 mM Rho-associated protein kinase (ROCK) inhibitor Y-27632
(Sigma-Aldrich) when iPSCs were thawed or passaged as single cells.

METHOD DETAILS

Mouse treatments
For glucose tolerance tests and in vivo glucose-stimulated insulin secretion, mice were intraperitoneally injected with 2 mg/g
dextrose (Sigma-Aldrich) after overnight fasting. For insulin tolerance tests, mice were intraperitoneally injected with 0.75 U/kg insulin
(Eli Lilly) after 6 hours of fasting. Tail vein blood glucose levels were measured using glucometers (Contour, Bayer). For Tamoxifen
treatment, mice were intraperitoneally injected once daily with either tamoxifen (TAM; 120 mg/kg body weight; Sigma-Aldrich)
dissolved in corn oil (Sigma-Aldrich) or vehicle for five consecutive days.

Cell and islet isolation

Cell sorting by flow cytometry was performed as described (Epshtein et al., 2017b). In brief, dissected pancreatic tissues of Nkx3-2-
Cre;R26-YFP mice were digested with 0.4 mg/ml collagenase P (Roche) and 0.1 ng/ml DNase (Sigma-Aldrich) diluted in HBSS for
30 min at 37 C with agitation. Digested pancreata were washed with FACS buffer (PBS Ca-/Mg- supplemented with 5% FBS and
5 mM EDTA), followed by cell filtration. Cells were blocked with goat serum (Sigma-Aldrich) for 30 minutes at 4 C followed by staining
with indicated antibodies for 30 minutes at 4 C. Cells were collected using FACSAria (BD Biosciences) when ECs were collected

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based on their PECAM1 expression and pericytes based on YFP expression. Dead cells were excluded using DAPI. Islets were iso-
lated according to standard protocols. In brief, collagenase P (0.8 mg/ml; Roche) dissolved in RPMI (Gibco) was injected through the
common bile duct into the pancreas of a euthanized mouse. Dissected pancreatic tissue was incubated for 11–15 min at 37  C, fol-
lowed by a gradient separation with Histopaque 1119 (Sigma-Aldrich) for 20 minutes at 1300 g, without brake. Islets were collected
from the gradient interface, followed by their manual collection.

In vitro glucose-stimulated insulin secretion

For mouse islets, groups of 10 isolated islets were washed and pre-incubated for 30 minutes in RPMI1640 (Glucose free; Gibco
#11879020) supplemented with 0.1% BSA and 25 mM HEPES containing 2.8mM glucose at 37 C. Islets were then incubated in either
2.8 or 16.7 mM glucose for 1 hour at 37  C, their supernatant collected, and secreted insulin levels measured.
For iPSC-derived b-like cells, five to six clusters (equivalent to 0.5–1.0 x 106 cells) were rinsed twice with Krebs buffer (129 mM
NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO2, 1 mM Na2HPO4, 1.2 mM KH2PO4, 5 mM NaHCO3, 10 mM HEPES, 0.1% BSA, in
deionized water and then sterile filtered) and then pre-incubated in Krebs buffer for 60 minutes at 37 C. Cells were then incubated in
Krebs buffer with 2 mM glucose for 60 minutes at 37 C. The cells were then transferred to another plate containing Krebs buffer with
20 mM glucose and incubated for additional 60 minutes at 37 C.

Measurement of hormones secretion and content

For serum hormone measurements, tail vein blood was collected in Microvette 100 Li-Hep tubes (Sarstedt; 201282), and serum was
separated after tubes were spun down for 5 minutes at 5000 g at RT. Pancreas and islet insulin contents were extracted by overnight
incubation in 1.5% HCl and 70% ethanol, followed by cell lysis using TissueLyser II (Qiagen). Hormone levels were determined using
mouse ultrasensitive Insulin (Alpco, 80-INSMSU-E01), STELLUX Chemo rodent insulin (Alpco, 80-INSMR-CH01), mouse proinsulin
(Alpco, 80-PINMS-E01), mouse glucagon (Alpco, 48-GLUHU-E01), and human Insulin (#10-1132-01; Mercodia) ELISA kits.

Western blot analysis

Isolated islets from groups of six mice were pooled, cultured for 2 hours in CMRL (Gibco) supplemented with 10% FBS and 1% Pen-
Strep, and homogenized in RIPA buffer (ThermoFisher Scientific). Protein concentrations were measured using Pierce BCA Protein
Assay Kit (ThermoFisher Scientific). Equal amounts of proteins were separated on 10% Mini-PROTEAN TGX Precast Protein Gels
(Bio-Rad) and transferred to a nitrocellulose membrane. The membrane was stained with primary antibodies, followed by incubation
with HRP-conjugated secondary antibodies (Abcam). Signal was detected using ECL reagents (Cell Signaling).

Gene expression
RNA was extracted from isolated tissue and cells using PureLink RNA mini and micro kit (Invitrogen), respectively, according to the
manufacturer’s protocol. For qPCR analysis, this was followed by cDNA generation using high capacity cDNA reverse transcription
kit (Applied Biosystems) and TaqMan and SYBR Green assays (Applied Biosystems) (Table S1) were used to determine expression
levels of analyzed genes, normalized to GAPDH and cyclophilin expression, respectively. When indicated, a previously published
RNAseq data (Sakhneny et al., 2018) (deposited in https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5325/) was used to
determine gene expression.

Islet BMP signaling inhibition

Islets isolated from six mice were pooled, and 50 islets were cultured in a single 96-well-plate well containing media (CMRL (Gibco)
supplemented with 10% FBS and 1% Pen-Strep) either supplemented with 500 nM of the BMPR1A inhibitor LDN193189 hydrochlo-
ride or non-supplemented for 48 hours.

Immunofluorescence
Dissected pancreatic tissues were fixed in paraformaldehyde (4%), followed by cryo- or paraffin- sectioning. For cryosectioning,
samples were equilibrated in 30% sucrose solution and embedded in OCT compound (Tissue-Tek). Sections were blocked with
CAS block (Invitrogen) for 1 hour at RT, followed by staining with primary antibodies ON at 4 C. Thereafter, sections were incubated
with secondary fluorescent antibodies (AlexaFluor; Invitrogen) for 1 hour at RT. Slides were mounted with Vectashield mounting me-
dium containing DAPI (Vector Laboratories). For paraffin-section, antigen retrieval was performed by boiling the slides in Citra buffer
(BioGenex). Images were acquired using BZ-9000 BioRevo (Keyence) and SP8 confocal (Leica) microscopes.

Morphometric analysis
For analysis of functional vasculature, fluorescein-labeled tomato lectin (1 mg/ml; Vector Laboratories, FL-1171) was injected intra-
venously and allowed to circulate for 5 minutes before the animal was euthanized. Cryo-sections, at least 100 mm apart, were stained
as indicated when 30-40 islets per mouse were analyzed. Islets were defined as insulin+ areas, and either lectin+ or NG2+ areas within
the islets were measured. To measure b-cell mass, paraffin-embedded tissue sections were immunostained for insulin and counter-
stained with HCS CellMask Deep Red Stain (Invitrogen) to label the whole tissue sections. For newborn pancreata, every fifth section
(20% of total tissue) was analyzed. For adult pancreata, sections at least 100 mm apart were analyzed. For each mouse, the acquired
insulin+ area was divided by the whole acquired pancreatic area (labeled by HCS CellMask Stain) and multiplied by the pancreas

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weight. For b/a cell ratio paraffin-embedded tissue sections were immunostained for insulin and glucagon. Sections at least 100 mm
apart were analyzed. For each mouse, the acquired insulin+ area was divided by the acquired glucagon+ area. For cell proliferation
analysis, paraffin-embedded tissue sections were immunostained for Ki67 to mark proliferative cells and insulin. Sections at least
50mm apart were analyzed when 300-350 insulin+ cells per mouse were analyzed. For each mouse, the number of Ki67+ insulin+ cells
were manually counted and divided by insulin+ cells number.
For iPSC-derived b-like cell quantification, cryo-sections of 3D cell clusters were immunostained with indicated antibodies and
counterstained with Hoechst. For each cluster, the number of positive cells was manually counted and divided by the total number
of cells within the cluster.
Images were acquired using Keyence BZ-9000 (Biorevo) and ZEISS LSM 70 confocal microscopes and analyzed using ImageJ
software (NIH).

Differentiation of human iPSCs into pancreatic b-like cells

Human iPSC lines [BIHi043-A (XM001)](Wang et al., 2018) differentiation was carried out following a 21-day protocol previously
described in (Cozzitorto et al., 2020; Russ et al., 2015). Briefly, iPSCs were dissociated using Accutase and seeded at a density
of 5.5 x106 cells per well in ultra-low attachment 6-well plates (Thermo Fisher Scientific) in E8 medium supplemented with 10 mM
ROCK inhibitor, 10 ng/ml Activin A (R&D Systems), and 10 ng/ml Heregulin (Peprotech). Plates were placed on an orbital shaker
at 100 rpm to induce sphere formation at 37 C in a humidified atmosphere containing 5% CO2. To induce definitive endoderm dif-
ferentiation, cell clusters were incubated in Day (D)1 medium [RPMI (Invitrogen) containing 0.2% FBS, 1:5000 ITS (Invitrogen), 100 ng/
ml Activin A, 50 ng/ml WNT3a] into low attachment plates. Subsequently, cell clusters were differentiated into b-like cells by exposure
to the appropriate media as previously published (Russ et al., 2015). 10 ng/ml rBMP4 was added to the culture at D17 of differenti-
ation for 48 hours.

QUANTIFICATION AND STATISTICAL ANALYSIS

Prism 8 and 9 (GraphPad Software) were used to conduct the statistical analysis of all data when all quantitative results were
assessed by unpaired, two-tailed Student’s t-test. Statistical outliers (a = 0.05) were excluded from analyses. For the mouse studies,
physiological and morphometric measurements were carried on in a blinded manner. For the iPSCs differentiation, three independent
experiments with three replicates were analyzed for gene expression, and two independent experiments with three replicates were
analyzed for GSIS. More statistical details of experiments can be found in the figure legends. P % 0.05 was considered statistically
significant.

e5 Developmental Cell 56, 1–9.e1–e5, October 11, 2021