Page 1 of 23 Diabetes

Hyperglycemia, reduced hematopoietic stem cells,

and outcome of COVID-19

Authors
Benedetta Maria Bonora1,2, Paola Fogar1, Jenny Zuin1, Daniele Falaguasta1, Roberta Cappellari1,
Annamaria Cattelan1, Serena Marinello1, Anna Ferrari1, Angelo Avogaro1, Mario Plebani1, Daniela
Basso1, Gian Paolo Fadini1,2

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Affiliations
1 Department of Medicine, University of Padova, 35128 Padua, Italy
2 Veneto Institute of Molecular Medicine, 35128 Padua, Italy

Correspondence to:
Prof. Gian Paolo Fadini, MD PhD
Department of Medicine, University of Padova,
Via Giustiniani 2, 35128 Padua, Italy
Phone +39 049 8214318
gianpaolo.fadini@unipd.it

Word count
Main 2011; abstract 200
References 25
The manuscript has an online supplement

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 Diabetes Page 2 of 23

Abstract

Admission hyperglycemia has emerged worldwide as a predictor of poor COVID-19 outcome. Hyperglycemia
leads to a defect in circulating hematopoietic stem/progenitor cells (HSPCs) which, in turn, predicts diabetic
complications. Here, we explored whether reduced HSPCs mediated at least part of the prognostic effect of
hyperglycemia on COVID-19 outcome. We found that patients with COVID-19 (n=100) hospitalized in a non-
intensive setting displayed dramatically (50-60%) reduced levels of HSPCs measured by flow cytometry as
CD34+, CD34+CD45dim or CD34+CD133+ cells, compared with controls (n=595). This finding was highly
significant (all p<10-10) after multivariable adjustment, or manual 1:1 patient match, or propensity score

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matching. Admission hyperglycemia (≥7.0 mmol/l) was present in 45% of patients, was associated with a
significant further ~30% HSPCs reduction and predicted a 2.6-fold increased risk of the primary outcome of
adverse COVID-19 course (admittance to the intensive care unit or death). Low HSPCs were also associated
with advanced age, higher peak C-reactive protein and neutrophil/lymphocyte ratio. Independently from
confounders, one standard deviation lower CD34+ HSPCs was associated with a >3-fold higher risk of adverse
outcome. Upon formal analysis, reduction of HSPCs was a significant mediator of the admission
hyperglycemia on COVID-19 outcome, being responsible for 28% of its prognostic effect.

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Page 3 of 23 Diabetes

Diabetes is a major determinant of poor COVID-19 outcomes, including admittance to the intensive care unit
(ICU) and death (1). Despite the prevalence of diabetes among people with COVID-19 may not be higher than
expected (2), admission hyperglycemia has been reported in up to 50% of patients (3). Hyperglycemia results
from the hyper-inflammatory state of COVID-19 (4) in conjunction with possible beta-cell infection by SARS-
CoV-2 (5). Admission hyperglycemia even in individuals without a history of diabetes predicts adverse
COVID-19 course (6; 7) but the reasons for this association are unclear. Hyperglycemia may reflect a more
severe inflammatory process or a pre-existing propensity to metabolic dysregulation. On the other side,
hyperglycemia can compromise the function of immune cells and affect hematopoietic responses. Indeed,
diabetes impacts bone marrow (BM) function, leading to a shortage of circulating hematopoietic
stem/progenitor cells (HSPCs) (8). In humans and rodents with diabetes, remodeling of the BM niche hampers

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the physiologic mobilization of HSPCs into the bloodstream (9). In turn, low circulating HSPCs predict adverse
outcomes, including diabetic complications (10; 11) cardiovascular events, and death (12). The mechanistic
link between hyperglycemia and shortage of circulating HSPCs relies on excess myelopoiesis (13). Though
COVID-19 is characterized by exaggerated myelopoiesis (14), little is known about HSPC levels and their
clinical meaning in COVID-19.
Here, we hypothesize that hyperglycemia in COVID-19 affects circulating HSPCs and that pauperization of
HSPCs in turn mediates the adverse effect of hyperglycemia on COVID-19 outcomes.

Research Design and Methods

Study participants. This prospective observational study was conducted according to the principles of the
Declaration of Helsinki and the protocol was approved by the Ethical Committee of the University Hospital of
Padua (#4930/Ao/20). Patients were admitted between December 2020 and March 2021 to the Infectious
Disease ward of the same hospital and signed informed consent. Inclusion criteria were: age ≥18; symptomatic
PCR-confirmed SARS-CoV-2 infection. Exclusion criteria were: age >100; severe renal or liver disease;
advanced cancer; short life expectancy; pregnancy or lactation; inability to provide informed consent. We
recorded the following information: age, sex, cardiovascular risk factors (diabetes, obesity, hypertension,
smoking), comorbidities (cardiovascular disease, chronic kidney disease, cancer), pre-hospital medications,
laboratory data.
The control population was composed of individuals not affected by COVID-19, selected among those
attending the same hospital in prior years and who had the same measure of circulating HSPCs.

Measures and outcomes. HSPCs were defined by expression of CD34 with/without CD45-diminished
criterion and CD133 co-expression. Total CD34+, CD34+CD45dim and CD34+CD133+ HSPCs were quantified
in fresh peripheral blood as previously described (10), and reported as relative (/106 blood cells) or absolute
(/mL of blood) count (Figure S1).
The primary endpoint of the outcome analysis was the composite of ICU admittance or in-hospital death. The

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 Diabetes Page 4 of 23

secondary outcome was death at 6 months. Outcomes were ascertained by accessing the patients’ electronic
medical records and the local death registry.

Statistical analysis. Continuous data are presented as mean (SD) and categorical variables as percentages.
Comparisons between two groups were performed using Student’s t test for continuous variables or 2 for
categorical variables. Linear correlations were analyzed with Pearson’s r coefficient. Outcome analysis was
performed using logistic regression.
We used three approaches to compare COVID-19 patients with controls: i) retained the entire population and
adjusted the differences with multiple linear regression; ii) manually matched 100 patients for age, sex and
metabolic status (obesity and diabetes), further adjusting for residual confounders as above; iii) performed a

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1:1 propensity score matching (PSM) with the nearest neighbor method without replacement and 0.5 SD
caliber. Missing variables were handled with multiple imputation by chained equation using the decision tree
regressor method and the most frequent value as starting point. The between-group balance was good in the
absence of significant differences at p<0.05 or absolute standardized mean difference (SMD)<0.10.
To evaluate whether HSPCs mediated the association between hyperglycemia and adverse outcome, we used
the SPSS macro PROCESS (model 4) with 95% confidence interval from 5000 bootstrap replicates.

Results

Patients and controls. We included 100 patients with COVID-19 initially hospitalized in a non-intensive
care setting (Table 1). They were on average 67.8-year-old, 59% males and 75% had at least one co-morbid
condition. Despite only 18% of patients had pre-existing diabetes, 45% had admission hyperglycemia defined
as a first fasting plasma glucose (FPG) ≥126 mg/dl (7 mmol/l). Of those without a history of diabetes but with
admission hyperglycemia, only 17% had HbA1c ≥6.5% (48 mmol/mol).

Profoundly reduced HSPCs in patients with COVID-19. COVID-19 patients were compared with 595
controls (Table S1). After adjusting for confounders (age, sex, smoking, hypertension, cardiovascular disease,
chronic kidney disease, and medications), levels of all HSPC phenotypes were significantly ~50-60% lower in
COVID-19 patients compared to controls. Results were similar when comparing COVID-19 patients with 100
manually matched controls (Figure 1A) and comparing 87 COVID-19 with 87 controls matched using PSM
(Figure 1B). A profound and significant (always p<0.001) reduction of circulating HSPCs was found based on
expression of CD34 with/without the CD45-diminished criterion and CD133 co-expression and based on
absolute or relative cell count. For further analyses, we focused on CD34+ cells as the most reliable HSPC
phenotype (10).

HSPCs and features of COVID-19 patients. Levels of CD34+ HSPCs inversely correlated with age (r=-
0.23; p=0.02), peak CRP (r=-0.21; p=0.035), and admission FPG (r=-0.21; p=0.036), but not with diabetes

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Page 5 of 23 Diabetes

history or HbA1c. Patients with admission hyperglycemia had significantly lower HSPCs using any antigenic
definition and cell count (Figure 1C & S2). Among other hematological parameters, CD34+ HSPCs were
inversely correlated with white blood cell count (r=-0.23; p=0.018) and with the neutrophil-to-lymphocyte
ratio (N/L; r=-0.44; p<0.001), a marker of myeloid-biased hematopoiesis (Figure 2A). Age and FPG were the
non-hematologic features most consistently correlated with absolute and relative levels of all HSPC
phenotypes (Figure 2B).

HSPCs and COVID-19 outcomes. Most patients (87%) had pneumonia and the standard in-hospital
treatment included glucocorticoids and low-molecular weight heparin. FPG and HSPCs measured upon
hospitalization did not differ between patients who had started glucocorticoids at home (29%) compared to

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those who were not on glucocorticoids upon admission (FPG 129.4±39.8 vs. 143.8±73.3 mg/dl; p=0.317.
CD34+ HSPCs 138.2±104.7 vs. 167.8±113.0/106; p=0.229).
During the hospital stay (mean duration 15.7 days), 28 patients were admitted to the ICU or died. Patients with
such poor outcome (Table 1) had a higher prevalence of pre-existing CHD and CKD (reflected by lower
admission eGFR). They also exhibited higher admission FPG (175.4±81.1 vs. 125.8±52.7 mg/dl; p<0.001).
HSPC levels were significantly and further reduced in patients with poor compared to those with good COVID-
19 outcome (CD34+ 99.1±13.1 vs. 182.6±115.6 cells/106; p<0.001; Figure 1D & S3). The hospital stay was
longer for patients with poor outcome, who showed higher peak CRP and more often required high-flow
oxygen.
In logistic multivariable analyses (Table S2), CD34+ HSPC levels remained significantly associated with
adverse outcome in model 1 (adjusted for age and sex), model 2 (further adjusted for confounders at baseline,
i.e. CHD, eGFR and FPG), and model 3 (further adjusted for peak CRP, N/L, and high-flow oxygen). In the
fully adjusted model, 1 SD decrease in CD34+ HSPCs had an odds ratio for poor outcome of 3.1 (95% C.I.
1.08-8.92; p=0.036). Results were similar when HSPCs were defined as CD34+CD45dim cells in place of total
CD34+ cells (OR 3.1; 95% C.I. 1.10-8.77; p=0.033).
At 6 months after hospitalization, 17 patients had died. Relative counts of CD34+, CD34+CD45dim or
CD34+CD133+ HSPCs were significantly lower in those who died than in those who were alive at 6 months
(Figure 1E & S4).

Hyperglycemia, HSPCs and COVID-19 outcomes. Patients with admission hyperglycemia had a 2.58-
times higher rate of adverse outcome compared to those with FPG<7.0 mmol/l (42.2% vs. 16.4%; p=0.004).
Admission FPG was a predictor of poor COVID-19 outcome after adjusting for age, sex and baseline co-
variates (CHD and eGFR). However, when the CD34+ HSPC level was entered in the model, the predictive
value of FPG was diminished and no longer significant (Table S3). According to a mediation analysis, the
indirect effect of FPG on COVID-19 outcome mediated by CD34+ HSPCs was 27.9% of the total effect
(bootstrapped 95% C.I. 6.3% to 72.1%; p=0.01; Figure 3).

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 Diabetes Page 6 of 23

Discussion

Building from our previous studies on diabetes, we show that reduced HSPC levels are significant mediators
of the negative prognostic effect of hyperglycemia on COVID-19 outcome.
Recently, HSPCs from COVID-19 patients have been found stuck in the G1 phase and prone to apoptosis (14).
Furthermore, binding of the spike protein to ACE2 impaired HSPC survival and fostered myeloid
differentiation (15). We now show that COVID-19 leads to a dramatic pauperization of circulating HSPCs,
which is strongly and independently associated with adverse outcome.
Diabetes is a predictor of death from COVID-19 (2), but hyperglycemia is often observed even in non-diabetic
individuals with severe COVID-19 (6). Hyperglycemia exerts negative effects on innate immunity, favoring

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severe infections and increasing mortality from infectious diseases (16). Mechanistically, high glucose
modifies the activation of monocyte-macrophages, facilitating SARS-CoV-2 replication, suppressing T-cell
responses, and exacerbating lung damage in COVID-19 (17). Hyperglycemia also causes exaggerated
myelopoiesis (13) that typically elevates N/L, a feature of severe COVID-19 (16). Such excess myelopoiesis
hampers mobilization of HSPCs from the BM to peripheral blood (9), reducing circulating HSPCs (18). Indeed,
we found that hyperglycemia was a significant determinant of HSPC levels among COVID-19 patents. Despite
the correlation of HSPCs with glucose levels was relatively weak, it was similar to that with age, which is well
established. On the other side, the lack of correlation with HbA1c is consistent with the transient nature of
COVID-19-associated hyperglycemia (19). The stronger association of HSPCs with inflammatory markers
than with organ-damage markers supports that hyper-inflammation was responsible for reducing HSPCs.
Remarkably, entering HSPCs in the outcome model nullified the prognostic value of N/L, suggesting that the
link between N/L and severe COVID-19 may be ascribed to reduced HSPCs.
The strong link between low HSPCs and adverse outcomes relies on their multifaceted role in optimizing
hematopoiesis, modulating immune-inflammation, as well as maintaining vascular homeostasis (8). It is
therefore not surprising that HSPC derangement contribute to COVID-19 worsening, which has prominent
immune-inflammatory and vascular manifestations (20). Our new data are consistent with the interpretation
that HSPC reduction mediates part of the effect of hyperglycemia on COVID-19 outcome. The statistical
mediation analysis formally supports such concept providing a quantitative estimate of the direct and indirect
effects.
Though glucocorticoid therapy may confound the association between hyperglycemia and COVID-19
outcomes (21), in-hospital use of glucocorticoids was unlikely to affect our results because we measured FPG
and HSPCs upon admission. Additionally, we observed no difference in FPG and HSPC levels between
patients who initiated glucocorticoids at home or at time of hospitalization, ruling out a meaningful
confounding.
As a therapeutic implication of our findings, we speculate that the observed benefit of DPP-4 inhibitors against
COVID-19 progression (22) may rely on their well-known stimulatory effect on circulating progenitors (23).
We finally report that low HSPCs at time of hospitalization for COVID-19 were associated with mortality at
6 months. While the study was underpowered to show an independent effect, we anticipate that reduced HSPCs

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Page 7 of 23 Diabetes

may play a role in long-COVID. The immune checkpoint PD-L1 appears to be deficient in HSPCs from
patients with diabetes (24), and PD-1 overexpression has been related to long-COVID (25). Future studies
should therefore evaluate whether the PD-1/PD-L1 axis links HSPC defects to the development of long-
COVID.
We acknowledge that we only measured HSPC levels but not their function, including differentiation capacity
and skewness. While reduced HSPC levels can reflect alterations in survival and traffic, functional assays
would enable a better understanding of HSPCs in COVID-19. In addition, while we quantified HSPC only
upon hospitalization, a time-course analysis may reveal more insight along the disease development and
progression. Finally, though backed by prior mechanistic studies, a causal relationship cannot be biologically
proven with mediation analysis and will require further investigation in vitro and in vivo.

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In conclusion, we describe a new pathway whereby metabolic dysregulation reflected by admission
hyperglycemia is associated with low circulating HSPCs among COVID-19 patients. Along with other culprits,
such a shortage of circulating HSPCs may contribute to the prognostic effect of hyperglycemia on COVID-19
outcome.

Acknowledgements. None.

Data availability. Original data associated with this article are available from the corresponding author upon
reasonable request.

Author contributions: Study design: AA, GPF, AC, MP, DB. Data collection and analysis: BMB, PF, JZ,
DF, RC, SM, AF, DB. Manuscript writing: BMB; GPF; PF, AA. Manuscript revision. JZ, DF, RC, AC, SM,
AF, MP, DB. All authors approved the final version of the manuscript.

Guarantor: GPF is the guarantor of this work.

Conflict of interest: The authors declare no conflict of interest.

Funding: Supported by the Department of Medicine, University of Padova (Fondazione CARIPARO,

COVIDIMED project) and by the Italian Ministry of Education, University and Research, 2020 PRIN call
(202077EYN7). The study funders were not involved in the design of the study, collection, analysis, and
interpretation of data, writing the report, and imposed no restrictions regarding publication.

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Figure legends

Figure 1. Levels of circulating HSPCs in COVID-19 and clinical outcomes. A) Comparison of the
levels of CD34+ circulating hematopoietic stem/progenitor cells (HSPCs) in patients with coronavirus disease
2019 (COVID-19) and in control individuals. All controls are shown along with a subset of controls manually
matched for age, sex and metabolic status (see text). The bottom part of panel A shows the adjusted differences
in HSPC levels between COVID-19 and the two groups of control individuals (for adjusted co-variates see
text and tables). B) Comparison of the levels of circulating HSPCs in propensity score matched cohorts of
patients with COVID-19 and controls. The bottom part of panel B shows the difference in HSPC levels

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between COVID-19 and matched control individuals (no adjustment). C) Levels of CD34+ HSPCs in COVID-
19 patients with and without hyperglycemia defined using the standard 7.0 mmol/l threshold for fasting plasma
glucose (FPG). D) Levels of CD34+ HSPCs in COVID-19 patients experiencing poor (admittance to the
intensive care unit or death) or good outcome during hospitalization. E) Levels of CD34+ HSPCs in COVID-
19 patients according to their status (live/dead) at 6 months after hospitalization. Columns height represents
mean and bars represent standard error. Superimposed data points are referred to individual patients. P-values
are shown for the between-group comparisons.

Figure 2. Clinical correlates of HSPC levels in COVID-19. A) The strongest correlations for CD34+
HSPCs are shown as scatter plots. Patients are divided according to good or poor outcome (admittance to the
intensive care unit or death) using the color code described in the legend. Pearson’s r coefficients and the
respective p-values are shown. B) The heatmap shows values of Pearson’s r coefficients for each antigenic
definition of hematopoietic stem/progenitor cells HSPCs and their relative and absolute cell count in peripheral
blood. The code color allows easy interpretation: intense blue represents strong direct correlation, intense red
represents strong inverse correlation, and white represents no correlation. Cell boxing indicates statistical
significance at p<0.05.

Figure 3. Results of the mediation analysis. Panel A shows that standard model 4 for mediation analysis
using the PROCESS macro, which was implemented in SPSS: the effect of X onto Y is analysed through the
mediating effect of M. Panel B shows results of the analysis, were X is 1 mmol/l increase in admission fasting
plasma glucose (FPG), Y is adverse COVID-19 outcome (admittance to the intensive care unit or death), and
M is one standard deviation (SD) decrease in circulating CD34+ hematopoietic stem/progenitor cells (HSPCs).
Coefficients are shown for each connection in the model along with calculation of the percentage mediated
effect over the total effect. C) Graphical representation of the mediation effect.

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 Diabetes Page 12 of 23

12

Table 1. Clinical characteristics of COVID-19 patients. Patients were divided according to the
occurrence of the primary outcome during hospitalization. Data are expressed as mean (standard deviations)
or percentages.

Good Poor
Variable All p
outcome outcome
Demographics and anthropometrics
Number 100 72 28 -
Age (years) 67.8 (17.7) 66.9 (18.6) 70.3 (15.3) 0.393
Sex male (%) 59.0 59.7 57.1 0.816

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Comorbidities
Diabetes (%) 18.0 13.9 28.6 0.088
Hypertension (%) 54.0 50.0 64.3 0.202
Current Smoking (%) 8.0 9.7 3.6 0.418
Obesity (%) 18.0 15.3 25.0 0.260
CHD (%) 13.0 6.9 28.6 0.004
Atrial Fibrillation (%) 12.0 12.5 10.7 0.807
CKD (%) 13.0 5.6 32.1 <0.001
COPD (%) 6.0 6.9 3.6 0.528
Stroke (%) 8.0 6.9 10.7 0.537
COVID pneumonia (%) 87.0 83.3 96.4 0.082
GI symptoms, % 11.0 8.3 17.9 0.175
COVID-19 therapies
Low-flow oxygen (%) 46.0 59.7 10.7 <0.001
High-flow oxygen (%) 43.0 25.0 89.3 <0.001
Non-invasive ventilation (%) 28.0 13.9 64.3 <0.001
Invasive ventilation (%) 21.0 0.0 44.0 <0.001
Remdesevir (%) 43.0 41.7 46.4 0.670
Glucocorticoids (%) 87.0 84.7 92.9 0.282
Tocilizumab (%) 2.0 1.4 3.6 0.489
LMWH (%) 95.0 93.1 100.0 0.156
Convalescent plasma (%) 35.0 36.1 32.1 0.712
Medication before hospitalization
ACE inhibitors (%) 24.0 26.4 17.9 0.375
ARBs (%) 16.0 13.9 21.4 0.361
Calcium channel blockers (%) 19.0 18.1 21.4 0.703
Anti-platelet agents (%) 23.0 18.1 35.7 0.060

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Page 13 of 23 Diabetes

13

Statins (%) 27.0 23.6 35.7 0.225

Warfarin (%) 7.0 6.9 7.1 0.972
New anticoagulants (%) 8.0 5.6 3.6 0.686
Insulin (%) 8.0 1.4 25.0 <0.001
Other diabetes medications (%) 10.0 11.1 10.7 0.955
Laboratory
FPG, mmol/mol 7.7 (3.7) 7.0 (2.9) 9.7 (4.5)
<0.001
FPG, mg/dl 139.1 (66.1) 125.8 (52.7) 175.4 (81.1)
HbA1c, mmol/mol 44.4 (11.6) 43.0 (9.3) 47.6 (16.0) 0.086

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Serum creatinine, µmol/l 104.8 (122.4) 90.8 (114.4) 141.4 (136.1) 0.063
eGFR, ml/min/1.73 m2 76.6 (29.1) 82.7 (24.9) 60.9 (33.5) 0.001
C-reactive protein, mg/l 93.8 (7.7) 73.8 (60.7) 146.0 (88.9) <0.001
AST, U/L 40.0 (24.6) 38.7 (24.0) 44.3 (26.3) 0.307
ALT, U/L 41.1 (51.3) 35.5 (32.5) 45.0 (63.3) 0.329
LDH, U/L 356 (448.7) 334.6 (508.7) 424.2 (218.2) 0.371
Outcomes
Primary outcome (%) 28.0 0.0 100 -
Death (%) 13.0 0.0 46.4 -
ICU admittance (%) 21.0 0.0 75.0 -
Mean days of Hospitalization (%) 15.6 (12.8) 12.3 (8.3) 24.7 (17.3) <0.001

CHD, coronary heart disease; CKD, chronic kidney disease; COPD, chronic obstructive pulmonary disease;
GI, gastro-intestinal; LMWH, low molecular weight heparin; ACEi, ACE inhibitors; ARB, Angiotensin
receptor blockers; GLM, glucose-lowering medication; FPG, fasting plasma glucose; eGFR, estimated
glomerular filtration rate (CKD-EPI equation); AST, aspartate aminotransferase; ALT, alanine
aminotransferase; LDH, lactate dehydrogenase; ICU, intensive care unit.

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 Diabetes Page 14 of 23

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Figure 1. Levels of circulating HSPCs in COVID-19 and clinical outcomes. A) Comparison of the levels of
CD34+ circulating hematopoietic stem/progenitor cells (HSPCs) in patients with coronavirus disease 2019
(COVID-19) and in control individuals. All controls are shown along with a subset of controls manually
matched for age, sex and metabolic status (see text). The bottom part of panel A shows the adjusted
differences in HSPC levels between COVID-19 and the two groups of control individuals (for adjusted co-
variates see text and tables). B) Comparison of the levels of circulating HSPCs in propensity score matched
cohorts of patients with COVID-19 and controls. The bottom part of panel B shows the difference in HSPC
levels between COVID-19 and matched control individuals (no adjustment). C) Levels of CD34+ HSPCs in
COVID-19 patients with and without hyperglycemia defined using the standard 7.0 mmol/l threshold for
fasting plasma glucose (FPG). D) Levels of CD34+ HSPCs in COVID-19 patients experiencing poor
(admittance to the intensive care unit or death) or good outcome during hospitalization. E) Levels of CD34+
HSPCs in COVID-19 patients according to their status (live/dead) at 6 months after hospitalization. Columns
height represents mean and bars represent standard error. Superimposed data points are referred to
individual patients. P-values are shown for the between-group comparisons.

138x134mm (1000 x 1000 DPI)

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Page 15 of 23 Diabetes

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Figure 2. Clinical correlates of HSPC levels in COVID-19. A) The strongest correlations for CD34+ HSPCs are
shown as scatter plots. Patients are divided according to good or poor outcome (admittance to the intensive
care unit or death) using the color code described in the legend. Pearson’s r coefficients and the respective
p-values are shown. B) The heatmap shows values of Pearson’s r coefficients for each antigenic definition of
hematopoietic stem/progenitor cells HSPCs and their relative and absolute cell count in peripheral blood. The
code color allows easy interpretation: intense blue represents strong direct correlation, intense red
represents strong inverse correlation, and white represents no correlation. Cell boxing indicates statistical
significance at p<0.05.

171x96mm (1000 x 1000 DPI)

For Peer Review Only

 Diabetes Page 16 of 23

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Figure 3. Results of the mediation analysis. Panel A shows that standard model 4 for mediation analysis
using the PROCESS macro, which was implemented in SPSS: the effect of X onto Y is analysed through the
mediating effect of M. Panel B shows results of the analysis, were X is 1 mmol/l increase in admission
fasting plasma glucose (FPG), Y is adverse COVID-19 outcome (admittance to the intensive care unit or
death), and M is one standard deviation (SD) decrease in circulating CD34+ hematopoietic stem/progenitor
cells (HSPCs). Coefficients are shown for each connection in the model along with calculation of the
percentage mediated effect over the total effect. C) Graphical representation of the mediation effect.

171x122mm (1000 x 1000 DPI)

For Peer Review Only

Page 17 of 23 Diabetes

ONLINE SUPPLEMENT

Table S1. Comparison between COVID-19 and control patients. Key clinical characteristics of
patients with COVID-19 are compared to those of control patients. Three groups of controls were considered
as described in the text. For the propensity score matched groups, in addition to p-values, absolute standardized
mean differences (SMD) are shown.

All Controls Propensity score matching

COVID-19 All p Manual match p COVID-19 Controls p SMD

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Number 100 595 100 87 87
Age, years 67.8 (27.6) 52.2 (16.3) <0.001 67.3 (13.7) 0.802 64.8 (16.9) 63.0 (17.7) 0.475 0.109
Male sex, % 59.0 46.6 0.021 59.0 1.000 59.8 57.5 0.760 0.033
Diabetes, % 18.0 15.3 0.492 18.0 1.000 20.7 14.9 0.325 0.107
Smoking, % 8.0 18.0 0.015 8.0 1.000 8.0 12.6 0.376 0.107
Obesity, % 18.0 12.1 0.104 18.0 1.000 19.5 16.1 0.555 0.064
Hypertension, % 54.0 25.4 <0.001 58.6 <0.001 50.6 47.1 0.651 0.049
Cardiovascular disease, % 20.0 6.7 <0.001 26.7 0.030 18.4 16.1 0.690 0.043
Chronic kidney disease, % 13.0 7.2 0.050 18.3 0.272 13.8 13.8 1.000 0.000
Anti-platelet agents, % 23.0 12.8 0.007 37.1 <0.001 23.0 24.1 0.859 0.019
RAS blockers, % 39.0 16.1 <0.001 40.0 <0.001 35.6 35.6 1.000 0.000
Statin, % 27.0 11.8 <0.001 30.0 0.097 25.3 23.0 0.725 0.038
HSPC phenotype
CD34+ /106 159.2 (196.2) 426.5 (196.0) <0.001* 400.8 (289.5) <0.001# 156.5 (110.1) 396.1 (198.4) <0.001 -
CD34+CD45dim /106 120.3 (179.0) 284.2 (179.0) <0.001* 319.4 (191.4) <0.001# 120.2 (103.1) 260.0 (152.1) <0.001 -
CD34+CD133+ /106 60.5 (101.6) 133.5 (101.6) <0.001* 159.5 (110.2) <0.001# 62.1 (66.2) 129.4 (77.7) <0.001 -
CD34+ / ml 1045.0 (1439.7) 2855.1 (1439.7) <0.001* 2781.9 (1500.3) <0.001# 1062.0 (918.9) 2648.3 (1182.4) <0.001 -
CD34+CD45dim / ml 807.8 (1452.4) 2277.5 (1452.4) <0.001* 2421.3 (1451.6) <0.001# 832.7 (860.6) 2075.2 (1145.0) <0.001 -
CD34+CD133+ / ml 430.1 (798.8) 1055.2 (798.8) <0.001* 1200.2 (824.6) <0.001# 454.6 (597.6) 1035.3 (591.8) <0.001 -

RAS, renin angiotensin system. * Adjusted for age, sex, smoking, hypertension, cardiovascular disease,
chronic kidney disease, and use of medications. # Adjusted for hypertension, cardiovascular disease, anti-
platelet agents, and RAS blockers.

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 Diabetes Page 18 of 23

Table S2. Outcome analysis. The association between one standard deviation (SD) change in CD34+
hematopoietic stem/progenitor cells (HSPCs) with the primary outcome (poor COVID-19 course, dependent
variable) was ascertained by means of logistic regression models adjusted for an increasing number of co-
variates. Model 1 was adjusted for age and sex. Model 2 was further adjusted for covariates at baseline and
run first without HSPCs (to show the effects of FPG) and then with HSPCs. Model 3 was fully adjusted for all
confounders. Odds ratios (OR) are shown along with 95% confidence intervals (C.I.).

Model 1 Model 2 without Model 2 with Model 3

HSPCs HSPCs

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Variable OR (95% C.I.) p OR (95% C.I.) p OR (95% C.I.) p OR (95% C.I.) p
Age (/10 years) 1.00 (0.76-0.13) 0.978 0.78 (0.54-1.13) 0.176 0.69 (0.46-1.04) 0.077 0.98 (0.92-1.03) 0.347
Sex (male vs. female) 0.94 (0.36-2.43) 0.895 1.00 (0.36-2.78) 0.993 1.00 (0.34-2.90) 0.994 0.55 (0.12-2.58) 0.452
CHD (yes vs. no) - - 2.37 (0.54-10.3) 0.251 2.40 (0.50-11.5) 0.271 3.03 (0.27-34.1) 0.370
eGFR (/10 ml/min/1.73 m2) - - 0.76 (0.62-0.95) 0.013 0.75 (0.59-0.75) 0.018 0.73 (0.53-1.01) 0.057
FPG (/mmol/l) - - 1.17 (1.01-1.35) 0.033 1.14 (0.98-1.32) 0.083 1.07 (0.86-1.34) 0.554
Peak CRP (/10 mg/l) - - - - - - 1.08 (0.99-1.19) 0.085
High-flow oxygen (yes vs. no) - - - - - - 27.7 (4.92-155) <0.001
N/L ratio (/unit) - - - - - - 0.99 (0.90-1.10) 0.859
CD34+ HSPCs (/SD) 0.30 (0.14-0.63) 0.002 - - 0.32 (0.14-0.73) 0.006 0.32 (0.11-0.93) 0.036

CHD, chronic heart disease; eGFR, estimated glomerular filtration rate, FPG, fasting plasma glucose; CRP, C-
reactive protein; N/L neutrophil/lymphocyte.

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Page 19 of 23 Diabetes

Table S3. Mediation analysis. The association between one mmol/l change in fasting plasma glucose
(FPG) and the primary outcome (dependent variable) was evaluated with and without

Model without HSPCs Model with HSPCs

Variable OR (95% C.I.) p OR (95% C.I.) p
Age (/ year) 0.78 (0.54-1.13) 0.176 0.69 (0.46-1.04) 0.077
Sex male 1.00 (0.36-2.78) 0.993 1.00 (0.34-2.90) 0.994
CHD (yes vs no) 2.37 (0.54-10.3) 0.251 2.40 (0.50-11.5) 0.271
eGFR (/ 10 ml/min/1.73 m2) 0.76 (0.62-0.95) 0.013 0.75 (0.59-0.95) 0.018
FPG (/ mmol/l) 1.17 (1.01-1.35) 0.033 1.14 (0.98-1.32) 0.083

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HSPCs (/ SD) - - 0.32 (0.14-0.73) 0.006

CHD, chronic heart disease; eGFR, estimated glomerular filtration rate. HSPCs, hematopoietic
stem/progenitor cells.

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 Diabetes Page 20 of 23

Figure S1. The gating strategy for quantification of HSPCs. Cells were labelled with the following
monoclonal antibodies: phycoerythrin (PE)-labelled anti-human CD34 (20 µl, BD Biosciences),
allophycocyanin (APC)-labelled anti-human CD133 (5 µl, Beckman Coulter), and Electron Coupled Dye
(ECD)-labelled anti-human CD45 (10 µl Beckman Coulter). After red blood cell lysis, the analysis was run on
a Beckman Coulter analyzer (NAVIOS EX Flow Cytometer) with a target of 106 total cytometric events (actual
average achieved 0.95 x 106). Data were analyzed by Kaluza software (Beckman Coulter). Different definitions
of HSPCs were used. After selecting the mononuclear cell fraction in the morphological side scatter versus
forward scatter gate, total CD34+ cells were quantified in the FSC vs CD34 fluorescence intensity plot for
comparability with previous studies and with the literature. Identity of CD34+ cells as HSPC was further
confirmed by low expression of CD45, thus identifying CD34+CD45dim cells. In addition, we determined co-

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expression of the stem cell marker CD133 con the CD34+CD45dim population (CD34+CD133+ HSPCs). The
same trained operator performed the analysis throughout the study. Relative cell count was expressed as cells
/ 106 events (WBC), while absolute cell count was reported per ml of blood. The absolute cell count was
obtained by multiplying relative cell count x WBC (white blood cell count, x103/mL).
Side scatter

CD34-PE

Forward scatter Side scatter

Gate on CD34+ cells
CD133-APC
CD45-ECD

Side scatter CD34-PE

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Page 21 of 23 Diabetes

Figure S2. Hyperglycemia and HSPCs in COVID-19. Levels of hematopoietic stem/progenitor cells
(HSPCs) are shown in COVID-19 patients divided between patients with and without hyperglycemia defined
based on the standard 7.0 mmol/l threshold for fasting plasma glucose (FPG). Data are presented for all the
three phenotypes expressed as absolute or relative cell counts in peripheral blood. Columns height represents
mean and bars represent standard error. Superimposed data points are referred to individual patients. P-values
are shown for the between-group comparisons.

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600 500
p=0.015 p=0.032
CD34+CD45dim / 106

500 CD34+CD133+ / 106

400
400
300
300
200
200

100 100

0 0
FPG (mmol/l) <7.0 7.0+ <7.0 7.0+

6000 6000 6000

p=0.004 p=0.005 p=0.016
CD34+CD45dim / mL

CD34+CD133+ / mL
CD34+ HSPCs / mL

5000 5000 5000

4000 4000 4000

3000 3000 3000

2000 2000 2000

1000 1000 1000

0 0 0
FPG (mmol/l) <7.0+ 7.0+ <7.0+ 7.0+ <7.0 7.0+

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 Diabetes Page 22 of 23

Figure S3. HSPC levels and in-hospital outcome of COVID-19. Levels of hematopoietic
stem/progenitor cells (HSPCs) are shown in COVID-19 patients divided based on the occurrence or not of the
primary endpoint of poor outcome, defined as admittance to the intensive care unit or death. Data are presented
for all the three phenotypes expressed as absolute or relative cell counts in peripheral blood. Columns height
represents mean and bars represent standard error. Superimposed data points are referred to individual patients.
P-values are shown for the between-group comparisons.

600 500
p=0.001 p=0.001
CD34+CD45dim / 106

CD34+CD133+ / 106

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500 400
400
300
300
200
200

100 100

0 0
Outcome Good Poor Good Poor

6000 6000 6000

p<0.001 p=0.026 p=0.082
CD34+CD45dim / mL

CD34+CD133+ / mL
CD34+ HSPCs / mL

5000 5000 5000

4000 4000 4000

3000 3000 3000

2000 2000 2000

1000 1000 1000

0 0 0
Outcome Good Poor Good Poor Good Poor

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Page 23 of 23 Diabetes

Figure S4. HSPC levels and vital status at 6 months. Levels of hematopoietic stem/progenitor cells
(HSPCs) are shown in COVID-19 patients divided based on vital status at 6 months after hospitalization for
COVID-19. Data are presented for all the three phenotypes expressed as absolute or relative cell counts in
peripheral blood. Columns height represents mean and bars represent standard error. Superimposed data points
are referred to individual patients. P-values are shown for the between-group comparisons.

600 500
p=0.038 p=0.027
CD34+CD45dim / 106

CD34+CD133+ / 106
500 400

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400
300
300
200
200

100 100

0 0
Status at 6 mo Alive Dead Alive Dead

6000 6000 6000

p=0.102 p=0.108 p=0.083
CD34+CD45dim / mL

CD34+CD133+ / mL
CD34+ HSPCs / mL

5000 5000 5000

4000 4000 4000

3000 3000 3000

2000 2000 2000

1000 1000 1000

0 0 0
Status at 6 mo Alive Dead Alive Dead Alive Dead

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